NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF MICROBIOLOGY WITH NANOTECHNOLOGY
E-LEARNING
CLASS
SUBJECT
: II B.Sc.
: DIPLOMA IN DIAGNOSTIC MICROBIOLOGY-I (BACTERIOLOGY&
SEROLOGY)
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UNIT – I
Selection, collection and transport of specimens – Blood, Urine, Sputum, CSF, Pus & Faeces –
transport media and storage. Microscopic examination of specimen for Bacterial pathogens –
simple, differential staining and motility.
PART A:
1. Which of the following extracellular enzymes produced by Group A streptococci is called
"spreading factor," an enzyme important in skin and soft tissue infection?
A. Streptokinase
B. Hyaluronidase
C. M Protein
D. Deoxyribonuclease C
E. None of the above
Correct Answer: D
2. All of the following statements about the M-protein of Group A Streptococci are correct
EXCEPT:
A. The amino terminal portion (distal portion) is variable, accounting for over 80 distinct
serotypes.
B. M proteins allow streptococci to resist phagocytosis.
C. Antibodies to M protein confer type-specific immunity.
D. M protein is the major virulence factor of Group A streptococci.
E. M protein is the major constituent of the capsule of Group A streptococci.
Correct Answer: E
3.A 12 year old boy presents with acute onset of sore throat, fever to 38.9 degrees C and painful
anterior cervical lymphadenopathy. On exam the pharynx is red and swollen and the tonsils are
covered with yellow-white exudate. The child also has halitosis. Which of the following nonsuppurative complications are of concern?
A. Sinusitis
B. Acute rheumatic fever alone
C. Acute glomerulonephritis alone
D. Acute rheumatic fever and acute glomerulonephritis
E. Scarlet fever alone
Correct Answer: D
4. Which of the following statements about Group B streptococci (Streptococcus agalactiae) is
not correct?
A. They are important causes of toxic strep syndrome.
B. They are frequent colonizers of the female genital tract.
C. Screening for this pathogen during pregnancy has reduced the incidence of neonatal sepsis.
D. These organisms are b-hemolytic.
E. They are important causes of urinary tract infections and bacteremia in elderly and diabetic
adults.
Correct Answer: A
5.Which of the following statements about the 23-valent pneumococcal vaccine is not correct?
A. It is a protein-conjugated, polysaccharide vaccine.
B. It is poorly immunogenic in young children and immunocompromised hosts.
C. It is routinely recommended for immune competent adults and children >2 yrs. of age at risk
for serious pneumococcal disease.
D. It protects against the major serotypes of pneumococci causing infection.
E. An adult with asplenia would be a candidate for this vaccine.
Correct Answer: A
PART B:
1.Write a note on Selection, collection & transport of sputum specimen from throat?
THROAT
 Possible pathogens
 Gram +: S.pyogenes, other β-hemolytic strep, C.diptheriae, C.ulcerans
 Gram -: Borellia vincenti, Leptotrichia buccalis, Bacteriodes melaninogenicus,
N.meningitis
THROAT
 Collection
 Patient mouth widely opened and tongue is depressed with sterile spatula
 Using sterile cotton swab gently rubbed against the lesion (grayish yellow mem –
Dip / tonsils – Strep)
 Avoid contamination with saliva
 Place the swab immediately in to sterile container & seal with adhesive tap.
 Submit the swab to lab
THROAT
Processing of throat specimen
o Sputum
 Possible pathogens
o G+
 Str.pneumoniae, Staph.aureus, Str.pyogenes
o G
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Kleb.pneumoniae, P.aeruginosa, Preteus sp, Yersinia pestis, H.influenzae.
Others
 M.tub, My.Pneu, Leg.Pneumophila
 Fungi: B.dermatitidis, H.capsulatum, A.fumigatus, C.albicans,
Cryp.neoformans, N.asteriods
 Sputum
Clean mouth with water
Collect directely in to petridish
Not expectorate- induct
10-15 % saline increase transudation and exfoliation
3 % for asthma patient
10 % PPG minimize aerosols
2.Explain in details about Selection, collection and transport of specimens – Blood, Urine,
Sputum, CSF, Pus & Faeces – transport media and storage.
COLLECTION, TRANSPORT AND EXAMINATION OF SPECIMEN
Criteria to be followed
 Type of specimen
 Time of collection
 Its collection methodology
 Method of its dispatch to the laboratory
Types of specimen
 Depend on the pathogen to be isolated
 For respiratory pathogen – sputum, not saliva
 N.gonorrhoeae – cervical swab, not vaginal swab
 Time of collection
 Depend on the pathogen to be isolated
 The condition of patient
 Times agreed between the medical, nursing and lab staff to deliver
Types of specimen
Urine – early morning
 Blood – Body temp begins to rise
 Precaution
 Ensure the site of collection
 A septic procedure – blood & CSF
 Avoid contamination – ulcer materials with skin commensals
 Swab free from antimicrobials
 Container – clean, sterile, leak proof, dry, free from disinfectant and easy to handle
 Transport
 Must be packed well and safely
 Precaution
 Keep register – record name, number, ward or health center of the patient, type of
specimen, investigation required, date of dispatch and the method of sending the
specimen
 Ensure – Specimen container free from cracks, leak proof cap
Transport
 Place the packed container in strong protective tin or box and seal completely
 Mark – specimens “High risk”
 Dispatch slides – box or envelope
 Label specimens – “Fragile with care – pathological specimen”
 Thermos containing ice cubes – for easily deteriorative specimens.
 Preservatives
 Urine - boric acid
 Sputum – Cetyl pyridinum chloride – NaCl solution
 Formaldehyde solution – must not be used for microbiological specimen
 Transport media
 Amies transport media – N.gonorrhoeae
 Cary – Blair medium - feces
 Refrigeration – 4 to 10 oC – rduce commensals multiplication
 Never refrigeration – Haemophilus, S.pneumoniae or Neiserria – cold kills these
pathogen
 Smear – place in dry, protected from dust, ants and flies.
THROAT
 Possible pathogens
 Gram +: S.pyogenes, other β-hemolytic strep, C.diptheriae, C.ulcerans
 Gram -: Borellia vincenti, Leptotrichia buccalis, Bacteriodes melaninogenicus,
N.meningitis
THROAT
 Collection
 Patient mouth widely opened and tongue is depressed with sterile spatula
 Using sterile cotton swab gently rubbed against the lesion (grayish yellow mem –
Dip / tonsils – Strep)
 Avoid contamination with saliva
 Place the swab immediately in to sterile container & seal with adhesive tap.
 Submit the swab to lab
THROAT
Processing of throat specimen
o Sputum
 Possible pathogens
o G+
 Str.pneumoniae, Staph.aureus, Str.pyogenes
o G Kleb.pneumoniae, P.aeruginosa, Preteus sp, Yersinia pestis, H.influenzae.
 Others
 M.tub, My.Pneu, Leg.Pneumophila
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Fungi: B.dermatitidis, H.capsulatum, A.fumigatus, C.albicans,
Cryp.neoformans, N.asteriods
 Sputum
Clean mouth with water
Collect directely in to petridish
Not expectorate- induct
10-15 % saline increase transudation and exfoliation
3 % for asthma patient
10 % PPG minimize aerosols
3. Write a nite on Sputum.
SPUTUM
 Preservetives
o Cetyl pyridinum chloride – NaCl sol for Mycobacterium tuberclosis
 Amies transport medium
 Cary-Blair medium for Y.pestis
 Sputum
 Workup Guidelines for Salmonella, Shigella, Aeromonas & Plesiomonas species
 Enteric Screening Procedure
o Conventional
 TSI or KIA, LIA, and Urea
o Commercial kits
 latex agglutination
 Full ID of suggestive screening results
 Serological identification of Salmonella and Shigella sp.
 Campylobacter sp.
 Most frequently isolated
 Selective media
o Campy-Thio (enrichment broth)
o Campy-BAP
o Skirrow medium
o Campylobacter-cefoperazone-vancomycin-amphotericin (CVA)
 Identification
o growth at 42°C, oxidase and catalase positive, Hippurate positive
o Nalidixic Acid susceptible, Cephalothin resistant
o Latex agglutination test
o Campylobacter sp.
o ProSpecT® Campylobacter Microplate Assay
o detects Campylobacter specific antigens in stool (fresh or in transport medium)
o utilizes polyclonal anti- Campylobacter specific antigens capture antibody
o can be read visually or spectrophometrically
o Evaluated in three studies
 Sensitivities of 80, 89 and 96%
 Specificities of 99%
o Flexiable, easy to use
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o reduces cost, reduce turnaround time
o Cross-reactivity with C. upsaliensis, C. hyointestinalis, or C. helveticus unknown
Genus Vibrio
Laboratory Diagnosis
o Media
 TCBS (thiosulfate citrate bile salts sucrose) agar
 sucrose-fermenting (Yellow colonies)
o V. cholerae, V. alginolyticus, & V. fluvialis
 non-sucrose-fermenting (green colonies)
o V. parahaemolyticus, V. vulnificus (Lactose Fermentor)
o Susceptible to 150 mg of vibriostatic agent (O/129)
Escherichia coli
Three paradigms by which diarrhea is produced:
o enterotoxin production
 Enterotoxigenic E. coli (ETEC)
 Enteroaggregative E. coli (EAEC)
o invasion
 Enteroinvasive E. coli (EIEC)
o intimate adherence with membrane signaling
 Enteropathogenic E. coli (EPEC)
 Enterohemorrhagic E. coli (EHEC/ STEC)
Enterohemorrhagic E. coli
Aka Shiga toxin-producing E. coli (STEC)
There are at least 100 serotypes of STEC
Only one serotype, namely E. coli O157:H7 can be detected in clinical laboratories.
o Selective media: sorbitol-MacConkey agar
o confirm by latex agglutination
Varied geographic distribution - evaluate prevalence for the need of routine workup
Availability of EIA for detection of STEC
Yersinia enterocolitica
Detection based on conventional methods
Selective media - CIN agar
o dark red “bull’s eye” with a transparent border
Clostridium difficile Testing
Acceptable specimen
o Unformed stool specimen unless ileus due to C. difficile is suspected.
Rejection criteria
o Specimens that are not liquid or soft
o Specimens from infants under 1 year old should be discouraged
o Specimen more than 24 hours old.
o Rectal swab specimens
o “Test for cure” or testing from asymptomatic individuals
Clostridium difficile Testing
Culture – most sensitive
o Selective medium - Cycloserine-cefoxitin-fructose agar
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o Characteristic horse-dung smell
o typical yellow-green fluorescence under UV light
o Limitations
 does not distinguish between toxigenic and non-toxigenigic strains
 delayed turn-around time
Use of latex agglutination test that detects glutamte dehydrogenase is discouraged
 Clostridium difficile Testing
Cell Culture Cytotoxicity Assay – most specific
o detects Toxin B
o Limitations
 Requires 24 to 48 hours
 Tedious
 Non-commercial versions are not standardized
EIAs for toxin A or toxins A and B
o Rapid
o Less sensitive than cell cyotoxicity assay
o Tests that detect only toxin A may miss isolates that are toxin A-B+
PART C:
Explain about specimen collection from Wound infection.
 WOUNDS
 Wound Cultures: Controversies
 Is sampling a wound for culture relevant?
 When and how should wounds be sampled?
 How should samples be transported?
 What analysis should be requested?
o Gram stain only? Culture only? Susceptibility testing?
o Quantitative cultures
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Wounds:Classification
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Acute
Caused by external damage to intact skin
Types
o Surgical
o Bites
o Burns
o Minor cuts
o Abrasions
o Severe traumatic
Chronic
Precipitated by predisposing conditions that lead to compromise of dermal/epidermal
tissue
Types
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o Impaired venous drainage
o Impaired arterial supply
o Metabolic diseases eg. diabetes
Wound Infections:Etiology
Surgical wounds
o Aerobes: S. aureus, coagulase negative staphylococci, Enterococcus spp. E. coli,
P. aeruginosa, Enterobacter spp.
o Anaerobes: Bacteroides spp., Peptostreptococcus, Clostridium spp.
Acute soft tissue infections
o Staph aureus only organism in 30%
o 30-50% mixed aerobes/anaerobes
o 20-30% other eg. Group A streptococci, Clostridium spp.
Bite wounds
o Special pathogens: Pasteurella multocida, Capnocytophaga canimorsus,
Bartonella henselae, Eikenella corrodens
o Other mixed aerobes and anaerobes
Wound Infections:Etiology
Burn wounds
o Primarily aerobic organisms: P. aeruginosa, Staphylococcus aureus, E. coli,
Klebsiella spp. Enterococcus spp. and Candida spp.
Diabetic foot ulcers
o Aerobes: Staph aureus, Streptococcus spp. P. aeruginosa, Enterococcus spp.,
enterics
o Anaerobes: Peptostreptococcus, Bacteroides spp., Prevotella spp.
Decubitus ulcers
o Mixed aerobic and anaerobic bacteria
o Wound Cultures
For open wounds
o Clean the wound margins with surgical soap or 70% ethyl or isopropyl alcohol
o Aspirate from the depth of the wound using a sterile syringe and needle
o Aspirated fluid should be sent to the laboratory in an appropriate transport system
o Alternatively, a curette may be used to obtain tissue from base of the wound
o Swabs are strongly discouraged
Wound Cultures
For closed wounds
o Prepare site as described for obtaining blood culture
o Aspirate as much purulent material as possible
o Transport in aerobic/anaerobic transport system
Wound Cultures: Gram stain
Pros
Useful in estimating organism load from tissue biopsies
Presence of microorganisms on smear from swabs correlates with > 106 organisms
(burns)
Facilitates identification of etiologic agent of wound infection following clean surgery
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Cons
Poor correlation seen between Gram stain and culture results from biopsy of diabetic foot
infections
In mixed infections, little value although presence of leukocytes indicates infection
Wound Specimens: Algorithms
Three approaches
o PMN predominance
o Q-Score
o Q-2-3-4 system
o Wound Specimens: Algorithms
Wound Cultures
Culture for aerobic and anaerobic bacteria if appropriately collected
o Gram stain results suggest adequate collection or presence of inflammation
o Tissues or aspirates vs. swabs
o Primary plating media: 5% SBA, Choc agar, MacConkey agar; anaerobic plates
and thio if appropriately collected
Identify anaerobes to Genus level only
Perform susceptibility testing of predominant organisms only
o Wound Cultures: Extent of Workup
Possible approaches
Use Gram stain result
o Work up organisms seen on stain only
o List others
Work up any potential pathogens to maximum of three, list others present by morphology
Work up any quantity S. aureus, P. aeruginosa, beta hemolytic streptococci, enterics and
gram-negative anaerobes
Wound Cultures: Examples
Gram stain results: (Acceptable)
Many neutrophils, no epithelial cells
Many gram positive cocci in clusters
Many gram negative bacilli
Few morphotypes resembling skin flora
Work up (identify and perform susceptibility testing): Gram positive cocci in clusters and
gram neg bacilli
Culture report: Many S. aureus, many Klebsiella pneumoniae, light aerobic bacteria
resembling skin flora
Wound Cultures: Examples
Gram stain: many neutrophils, few epithelial cells, Gram positive cocci in clusters, Gram
positive cocci in chains,
Culture grows: many S. aureus, many Group A streptococci, few enteric bacilli
Work up: S. aureus, Group A streptococcus: limited ID and no susceptibility on enteric
bacilli; susceptibility testing on Group A strep not required
Wound Cultures: Examples
Gram stain: Many neutrophils, few epithelial cells, multiple morphotypes
Culture grows: more than 3 potential pathogens
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Consider source
o Tissue or aspirate
o Contamination likely
o Type of patient
o May need to consult with clinician or Infectious Diseases service
o Q-Score
Workup of Wound Cultures
Q-Score System
o Good quality specimen (Q3)
 Up to 3 organisms can be considered as potential pathogens and worked
up (ID/AST)
o Lower quality specimen (Q2, Q1)
 More SEC
 Fewer organisms are worked up
 Workup of Wound Cultures
Q-Score System
o If the Q-score is greater than or equals the PP in culture
 Workup all potential pathogens
o If Q-Score is less than the PP in culture
 Look at the Gram stain
 Workup all PP that are seen on GS
 Morphologically ID others
 If all PP present on GS then only Morph ID all
Workup of Wound Cultures
Q/2-3-4 System
o Culture workup is based on the # of PP present
 2PP – ID/AST
 3PP
 Look at the Gram stain
o Workup two PP if they are seen on GS
o If all 3 present on GS then Morph ID
 4PP
 Morph ID only
o Wound Cultures: Example
Gram stain: many neutrophils, few epithelial cells, Gram positive cocci in clusters, Gram
positive cocci in chains,
Culture grows: many S. aureus, many Group A streptococci, few enteric bacilli
Q score = 2 [PMN (+3), few epi (-1)]
Q/2-3-4 = 3 PP
look at gram stain
Work up: S. aureus, Group A streptococcus, Morph ID and no susceptibility on enteric
bacilli
2. Explain about specimen collection from respiratory tract infection.
 RESPIRATORY SPECIMENS
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Respiratory Specimens
Upper respiratory tract specimens
o Throat
 detection of streptococcal pharyngitis
Respiratory Specimens
Upper respiratory tract specimens
o Nose
 detection of MRSA carriers
o Nasopharyngeal swabs
 diagnosis of Bordetella pertussis
o Nasopharyngeal swabs and washings
 diagnosis of viral disease
Lower Respiratory Tract Infections
“ The culture of lower respiratory specimens may result in more unnecessary
microbiologic
effort
than
any
other
type
of
specimen.”
Raymond C Bartlett
Lower
Respiratory
Tract
Infections
Epidemiology
Pneumonia is the sixth leading cause of death in US
Increasing numbers of patients at risk
o Aging population
o Increase in patients with immunocompromising conditions
Overtreatment has lead to resistance
o Multidrug resistant Streptococcus pneumoniae
o Resistance among hospital acquired pathogens such as Acinetobacter,
Pseudomonas aeruginosa and others
Cumitech
7B:2003
Lower Respiratory Tract Infections
6 contributing authors
Major sections
o Clinical aspects of diseases of LRT
o Specimen collection
o Specimen processing
o Interpretation of bacterial cultures
o Most common pathogens
o Methods for implementing change
o Guidelines for frequency of testing
o Public health issues
o Reimbursement codes
Categories of Lower Respiratory Tract Infections
Acute bronchitis
Community acquired pneumonia
Hospital acquired pneumonia
Pneumonia in the immunocompromised host
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Community
Acquired
Pneumonia
Etiologic Agents
Community
Acquired
Pneumonia
Diagnosis
Available Test Methodologies
Sputum Gram stain and culture
Blood cultures
Serologic studies
Antigen detection tests
Nucleic acid amplification tests
Sputum Gram Stain and Culture
Proponents
Demonstration of predominant morphotype on Gram stain guides therapy
Accuracy is good when strict criteria are used
Cheap, so why not?
Antagonists
Poor specimen collection
Intralaboratory variability (Gram stain interpretation)
Low sensitivity and specificity
Empiric treatment guidelines
Not cost effective
Sputum Collection
Proper patient instruction
o Food should not have been ingested for 1-2 h prior to expectoration
o The mouth should be rinsed with saline or water
o Patient should breathe and cough deeply
o Patient should expectorate into a sterile container
Transport container immediately to lab
Perform Gram stain and plant specimen as soon as possible
Sputum Gram Stain
Screen for acceptability
o Examine specimen under low power (x 10 objective)
o Examine 10 representative fields
o Specimens that show few squamous epithelial cells (< 10/lpf) and many PMNs (>
25/lpf) are acceptable
o Notify physician of unacceptable samples
Sputum
Gram
Stain
Unacceptable
Sputum
Gram
Stain
Good Quality
Sputum
Gram
Stain
Good Quality
Sputum Gram Stain
Good quality specimens
Quantify number and types of inflammatory cells
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Note presence of bronchial epithelial cells
Concentrate on areas with WBCs when looking for organisms
Determine if there is a predominant organism (> 10 per oil immersion field)
o Semiquantitate and report organism with descriptive
o If no predominant organism is present, report “mixed gram positive and gram
negative flora”
Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74.
Prospective study
Non immunocompromised patients hospitalized with CAP
1,000 bed hospital in Spain
ER physicians instructed on sputum collection for Gram stain and culture
Sputum collected under supervision of nurse or resident
o Samples were processed immediately
o Screened for epithelial cells
o Screened for predominant morphotype (> 75% of the organisms seen)
o Sputum planted to blood agar, chocolate agar and MacConkey agar
Strictly defined clinical and diagnostic parameters
Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74
Results
190/533 (35.6%) patients had no sputum sample submitted (these patients were included
in the calculations)
133/533 (25%) patients had a poor quality specimen
210/533 (39.4%) patients had a good quality specimen
Overall sensitivity and specificity for pneumococcal pneumonia: 57% and 97%
Overall sensitivity and specificity for H. influenzae pneumonia: 82 % and 99%
Gram stain gave presumptive diagnosis in 80% of patients who had a good specimen
submitted
> 95% of patients in whom a predominant morphotype was seen on Gram stain received
monotherapy
Gram Stain Reports
Be as descriptive as possible
o Moderate neutrophils
o Moderate Gram positive diplococci suggestive of Streptococcus pneumoniae
o Few bacteria suggestive of oral flora
Keep report short—avoid line listing of all morphotypes present
Sputum
and
Endotracheal
Suction
Culture Evaluation
Identify and perform susceptibility testing on 2-3 potential pathogens seen as
predominant on Gram stain
Alpha strep—rule out S. pneumoniae
Yeast—rule out Cryptococcus neoformans only
S. aureus, Gram negative bacilli
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o < normal flora, quantify and limit ID; no susceptibility
o Add comment that organism not predominant on stain
ID mould, Mycobacteria or Nocardia spp.
Modified from Sharp SE, et. Al. 2003. Cumitech 7B. ASM Press.
IDSA
Practice
Guidelines
Diagnostic Tests for CAP
Outpatients
o Empiric therapy with a macrolide, doxycycline, or a fluoroquinolone
Hospitalized patients with CAP
o Gram stain and culture of sputum
o 2 pretreatment blood cultures
o Studies for Mtb, Legionella in select patients
Rationale
o To improve patient care
o Advance knowledge of epidemiologically important organisms
o Prevent antibiotic abuse
o Reduce antibiotic expense
Bartlett JG. 2000. Clin Infect Dis 31:347-82.
ATS
Guidelines
Diagnostic Tests for CAP
Empiric therapy for outpatients
o Macrolide or tetracycline
Hospitalized patients with CAP
o 2 sets of pre-treatment blood cultures
o Pleural fluid Gram stain/culture when appropriate
o Studies for Legionella, Mtb, fungi in select patients
o Sputum Gram stain/culture only if resistant or unusual pathogen is suspected
o Avoid extensive testing
ATS. 2001. Am J Respir Crit Care Med 163: 1730-1754.
Hospital Acquired Pneumonia
Most frequent nosocomial infection (30-33% of cases) among combined medical surgical
intensive care units
83% are ventilator associated
Etiologic agents
Frequency (%)
o Gram positive cocci
 S. aureus
17
 S. pneumoniae
2-20
o Aerobic gram-neg bacilli
60
 Pseudomonas aeruginosa
 Enterobacter sp.
 Klebsiella pneumoniae
 Acinetobacter
 Legionella
o Anaerobes
10-20
o Fungi
0-10
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Modified from: Carroll KC. 2002. J Clin Microbiol 40: 3115-3120.
Hospital
Acquired
Pneumonia
Diagnosis
American College of Chest Physicians: Clinical findings are not sufficient for definitive
diagnosis
Qualitative culture or endotracheal sputum has poor predictive value
Bronchoscopy is recommended by many pulmonologists
o Bronchial brushings
o Bronchial washes
o Protected specimen brushing
o Bronchoalveolar lavage specimens (BAL)
o Transbronchial biopsy
o Respiratory Specimens
Protected Brush Specimen
o To procure uncontaminated lower airway secretions
o Brush within 2 catheters
Respiratory Specimens
Bronchoalveolar Lavage (BAL)
o Samples large area of the lung
o Performed using a bronchoscope
o 100 to 250 ml of saline injected
o Injected saline along with secretions is collected by aspiration
Transthoracic Aspiration
o Involves percutaneous introduction of a needle directly into the infiltrate
o Bronchoalveolar
Lavage
(BAL)
Specimen Acceptability
Microscopic examination of Gram-stained smear
o Acceptable
 <1% of cells present are squamous epithelial cells
o Unacceptable
 >1% of cells present are squamous epithelial cells
o Processing Bronchoscopy Specimens
Bronchoscopy brush protected
o Aerobic bacterial culture and Gram stain
o Anaerobic bacterial culture
o Limited volume
Bronchoscopy brush, unprotected
o No anaerobic culture
o Limited volume
Bronchial washings
o Useful only for pneumonia caused by strict pathogens
o Reasonable requests: Mtb, Fungi, Legionella, Pneumocystis
Bronchoalveolar lavage
o No anaerobe culture
o Amenable to extensive testing for all opportunistic pathogens
Interpretation of Quantitative PSB/BAL

















Dilution Method
o Quantify each morphotype present and express as CFU/ml
Calibrated Loop Method
o Quantify each morphotype present and express as log10 colony count ranges
Thresholds for significance
o PSB > 103 CFU/ml
o BAL > 104 CFU/ml
Baselski and Wunderink. 1994. Clin Micro Rev 7:547
Bronchoscopy
Samples
Quantitative Methods
PSB or BAL Baselski and Wunderink. 1994. Clin Micro Rev 7:546.
vortex 30-60 s
Final dilutions
Bronchoscopy
Samples
Quantitative Methods
Calibrated loop method
Baselski and Wunderink. 1994. Clin Micro Rev 7:547
o PSB
vortex 30-60 s BAL
Immunocompromised
Patients
Suggested BAL Protocol
Aerobic Gram stain quantitative bacterial culture
Fungal stain and culture
Mycobacterial stain and culture
Viral culture/Respiratory DFA
Pneumocystis DFA
Legionella culture
Genital Specimens
GENITAL TRACT SPECIMENS
 Patients in high risk situations:
 Patients known to have gonorrhea
 Male patients with NGU, PGU, epididymitis, and Reiter's Syndrome
 Females with mucopurulent cervicitis, urethral syndrome, endometriosis, and salpingitis
 Neonates born to infected mothers
 Infertility investigations
3. Explain in detail about specimen collection from Genital tract.
GENITAL TRACT SPECIMENS
 Sexually active asymptomatic females who:
 Are age 25 years or younger
 Are pregnant
 Have evidence of purulent or mucopurulent cervical discharge
 Exhibit endocervical bleeding, induced by swabbing on examination
 Have had a new sex partner in the preceding 2 months

Use no contraceptives or a non-barrier method for contraception
GENITAL TRACT SPECIMENS
 For Females
o Cervical specimens should be collected after removing excess mucous from the
cervical os and surrounding mucosa
o Use a second swab to collect specimen by rotating the swab for 10 to 30 secs. in
the endocervical canal
o Collect vaginal specimens using a speculum without any lubricant
GENITAL TRACT SPECIMENS
 For males
o Urethral specimens are collected by inserting a swab 2 to 4 cm. into the urethra
and rotating the swab for 2 to 3 seconds
 GENITAL TRACT SPECIMENS
 For HSV lesions
o Fluid from lesions should be aspirated using a syringe
o Swab can be used to collect vesicle fluid or cellular material from the base of the
lesion before crusting and healing have begun
 Genital Specimens
 LABORATORY DETECTION OF BV
 Clue Cells: vaginal epithelial cells studded with coccobacilli
o wet mount
 pH > 4.5
 Whiff test + (10-20% KOH)
 Scored gram stain
 Culture = NO
o G. vaginalis isolated in > 92% women with BV and 70% asymptomatic woman
 Probe: AFFIRM (Becton Dickinson)
o agrees well with high count G. vaginalis
 Trichomonas vaginalis
 Common sexually transmitted disease
 Disease associations and adverse outcomes
o Vaginitis
o Urethritis—men and women
o Outcomes
 Adverse pregnancy events
 Associated with increased HIV shedding
 Trichomonas
vaginalis
Diagnosis
 Culture- “gold standard”
o Diamond’s media
o InPouch TV; BioMed Diagnostics, San Jose CA)
 Barenfanger J, et. al. 2002. J Clin Microbiol 40:1387.
 Wet mount—insensitive (~ 50%)


















Rapid tests
o XenoStrip-Tv (GenzymeDiagnostics, Inc. San Antonio, Tex.)
 more sensitive than wet prep
 less sensitive than culture
 useful as a POC test
Pillay A, et. al. 2004. J Clin Microbiol 42:3853.
Kurth A, et. al. 2004. J Clin Microbiol 42:2940.
Lab detection (cont.)
GLC—no longer used
Detection of sialidases (neuraminidases that remove sialic acid from sialoglycoconjugates)
o In BV, associated with Prevotella and Bacteroides sp.
o Colorimetric test BVBlue System (Gryphus Diagnostics—91.7% sensitive; 97.8%
specific
Myziuk L, et. al. 2003. BVBlue test for diagnosis of bacterial vaginosis. J Clin Microbiol
41:1925.
Clue Cell of BV
BV
Scored
Gram
Stain
( from Nugent RP 1991;29:297)
Interpretation of Scored Gram Stain
0-3 = Normal
4-6 = Intermediate
o may indicate trichomoniasis, GC or CT
o abnormal gram stain, but not consistent with BV
7-10 = Consistent with Bacterial Vaginosis
o Significance of results unknown in pre-menarchal girls or post-menopausal
women
Who Should be Screened for BV?
Women with vaginal symptoms
o esp. if failed therapy
Pregnant women at high risk of preterm birth
Pregnant women with genital symptoms
o rule out trichomoniasis as well
Women with gynecologic surgery
4. Microscopic examination of specimen- Simple staining, Differential staining
& motility
 Smear & Fixation
 It is the process by which the internal & external struc of cells & mos are preserved ,
fixed in position.
 Mos is killed & attached firmly to slide during fixation.
 Two types of fixation
 Heat fixation
 Chemical fixation
 Heat fix- heat fix bacteria smear by gently flame heating & air dried film of bac.
 Chemical fix- penetrate cells & react with cellular comp- proteins, lipids to render them
inactive, insoluble, immobile
 Common fixative mixture contain ethanol, acetic acid, mercuric chloride &
formaldehyde.







Dyes
Two features
They have chromophore groups(conjugated with double bond)
They can bind with cells by ionic, covalent or hydrophobic bonding.
Ex. Positive dyes bind with negative cell structure.
Basic dyes
Acid dyes




Basic dyes- MB,basic fuchsin,CV,saf,MG- positive charge. (pentavalent nitrogen)
Basic dyes bind neg charge mol – nucleic acid, proteins, cell surface
Acid dyes- eosin, rose bengal, acid fuchsin- carboxyl & phenolic hydroxyls.
Bind positive cel
5. DIFFERENTIAL STAINING
Gram staining
 1884, Danish physician Christian Gram
 Its divides bacteria into gram negative, gram positive based on cell wall
 Primary stain
 Mordant stain
 Decolourizing agent
 Couter stain








Gram-stain Procedure
Acid fast staining
Another diff staining
Ziehl – Neelsen method
Basic fuchsin penetrate wuth the aid of heat & phenol
Cell wall have mycolic acid – a group of branched chain hydroxy lipids.
Acid fast cell are not easily decolourized by acid alcohol remain red colour.
Non acid fast – decolourized & stained with MB- blue colour.
Special staining
 Negative staining
 capsule staining
 Spore staining
 Flagella staining
 Nuclear staining
 Negative staining
India ink / Nigrosin dye (not heat fix)
 Spore staining (schaeffer Fulton procedure)
MG, Saf
veg – red colour
spore- green colour
 Negative staining
 Capsule staining
The Schaeffer-Fulton spore stain method
 Flagella Staining
 Thread like structure (10-30 nm) only seen under electron microscope.
 To observe with light microscope- flagella thickness by tannic acid and aluminum
potassium sulfate - mordant
 They stained with pararosaniline (leifson method) or basic fuchsin (gray method)
 Nuclear staining
 Smear
 Heat fix
 Dip in 1N HCl
 Water bath [60° C – 10min]
 Wash
 Flood Giemsa stain [2 -3 min]
 Wash
 Blot dry
UNIT – II
Cultivation and isolation of viable pathogens – Media used – differential, selective, enrichment
and enriched media.
PART A:
1.What is the O antigen of Enterobacteriaceae?
A. Cell surface polysaccharide
B. A channel controlling substance taken into the organism.
C. A flagellar protein
D. A peptidoglycan matrix important for cellular rigidity
E. Cell wall lipopolysaccharide
Correct Answer: E
2. All Enterobacteriaceae share all of the following characteristics EXCEPT:
A. Ferment glucose
B. Reduce nitrates to nitrites
C. Oxidase positive
D. Gram negative
E. Rod-shaped (bacilli)
Correct Answer:C
3. Which of the following virulence factors of E. coli is important for attachment to host
epithelial cells in the pathogenesis of urinary tract infections?
A. Aerobactin
B. Alpha hemolysin
C. Urease
D. K1 antigen
E. Pili
Correct Answer: E
4. This urinary pathogen "swarms" across agar surfaces and may cause bladder and renal calculi
(stones).
A. Citrobacter freundii
B. Enterobacter aerogenes
C. Serratia marcescens
D. Klebsiella oxytoca
E. Proteus mirabilis
Correct Answer: E
5. Which of the following statements regarding Enterotoxigenic E. coli are CORRECT?
A. They are important causes of traveler's diarrhea.
B. Transmission occurs from ingestion of contaminated food and water.
C. Disease is caused by production of one or both of two types of enterotoxins.
D. None of the above are correct.
E. All of the above are correct.
Correct Answer: E
PART B:
1.Cultivation and isolation of viable pathogens- Differential media;
Differential media or indicator media distinguish one microorganism type from another growing
on the same media.[5] This type of media uses the biochemical characteristics of a microorganism
growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin
y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a
microorganism. This type of media is used for the detection of microorganisms and by molecular
biologists to detect recombinant strains of bacteria.
Examples of differential media include:

blood agar (used in strep tests), which contains bovine heart blood that becomes
transparent in the presence of hemolytic Streptococcus

eosin methylene blue (EMB), which is differential for lactose and sucrose fermentation

MacConkey (MCK), which is differential for lactose fermentation

mannitol salt agar (MSA), which is differential for mannitol fermentation

X-gal plates, which are differential for lac operon mutants
Blood agar plate (BAP)
Blood agar plate (BAP) Contains mammalian blood (usually sheep or horse), typically at a
concentration of 5–10%. BAP are enriched, differential media used to isolate fastidious
organisms and detect hemolytic activity. β-hemolytic activity will show lysis and complete
digestion of red blood cell contents surrounding colony. Examples include Streptococcus
haemolyticus. α-hemolysis will only partially lyse (the cells are either lysed or not- it is the
digestion that may be incomplete) the hemoglobin and will appear green. An example of this
would be Streptococcus viridans. γ-hemolysis (or non-hemolytic) is the term referring to a lack
of hemolytic activity. Contains meat extract, tryptone, sodium chloride, and agar.
Chocolate agar
Chocolate agar (CHOC) is a type of blood agar plate in which the blood cells have been lysed by
heating the cells to 56 °C. Chocolate agar is used for growing fastidious (fussy) respiratory
bacteria, such as Haemophilus influenzae. No chocolate is actually contained in the plate; it is
named for the coloration only.
Horse blood agar
Horse blood agar (HBA) is a type of blood enriched microbiological culture media. As it is
enriched, it allows the growth of certain fastidious bacteria, and allows indication of haemolytic
activity in these bacterial cultures.
Thayer-Martin agar
Thayer-Martin agar (TM) is a chocolate agar designed to isolate Neisseria gonorrhoeae.
Thiosulfate-citrate-bile salts-sucrose agar
Thiosulfate-citrate-bile salts-sucrose agar (TCBS) enhances growth of Vibrio spp., including
Vibrio cholerae [5]
2.Cultivation and isolation of viable pathogens – Selective Media
A selective and differential media used to differentiate between Gram negative bacteria
while inhibiting the growth of Gram positive bacteria. The addition of bile salts and
crystal violet to the agar inhibits the growth of most Gram positive bacteria, making
MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose
fermenters, which form pink colonies, from lactose nonfermenters that form clear
colonies. An alternative media, eosin methylene blue (EMB) serves a similar purpose.

Mannitol salt agar (MSA)
MSA is also a selective and differential media. The mannitol indicates organisms that
ferment mannitol: mannitol fermentation produces lactic acid, lowering the pH and
turning the plate yellow. The salt is to select for halophiles; organisms that cannot
withstand a high salt content will be unable to grow well.

Mueller-Hinton agar (MHA)
MHA contains beef infusion, peptone, and starch and is used primarily for antibiotic
susceptibility testing. It can be in a form of blood agar.
Fungi growing in axenic culture (ascomycetes)
Aspergillus niger growing in potato dextrose agar

Xylose-Lysine-Deoxycholate agar (XLD)
XLD is used for the culture of stool samples and contains two indicators. It is formulated
to inhibit Gram-positive bacteria, while the growth of Gram-negative bacilli is
encouraged. The colonies of lactose fermenters appear yellow.
It is also used to culture possible Salmonella that may be present in a food sample. Most
Salmonella colonies will produce a black centre on XLD.
2.Explain General bacterial media& fungal media
Four types of agar plate demonstrating differential growth depending on bacterial metabolism

Bile esculin agar (BEA)
BEA is used for the isolation of Enterococci as well as Group D Streptococci

CLED agar (Cysteine Lactose Electrolyte Deficient agar)
CLED agar is used to isolate and differentiate urinary tract bacteria, since it inhibits
Proteus species swarming and can differentiate between lactose fermenters and nonfermenters.

Hektoen enteric agar (HEA)
HE agar is designed to isolate and recover fecal bacteria belonging to the
Enterobacteriaceae family. HE is particularly useful in isolating Salmonella and Shigella.


Lysogeny Broth (LB)

MacConkey agar (MAC)
Nutrient agar
Nutrient agar is usually used for growth of non-fastidious organisms and observation of
pigment production. It is safe to use in school science laboratories because it does not
selectively grow pathogenic bacteria.

Önöz agar
Önöz agar allows more rapid bacteriological diagnosis as Salmonella and Shigella
colonies can be clearly and reliably differentiated from other Enterobacteriaceae. The
yields of Salmonella from stool samples obtained, when using this medium, are higher
than those obtained with LEIFSON Agar or Salmonella–Shigella agar (SSA).

Phenylethyl alcohol agar (PEA)
PEA selects for Staphylococcus species while inhibiting Gram-negative bacilli (e.g.
Escherichia coli, Shigella, Proteus, etc.).

R2A Agar (R2A)
A non-specific agar that imitates the medium of water. Used for water analysis.
 Tryptic (Trypticase) Soy Agar (TSA)
TSA is a general purpose media produced via enzymatic digestion of soybean meal and
casein. TSA is frequently the base media of other agar types; for example, blood agar
plates are made by enriching TSA plates with blood (see above).
TSA plates support growth of many semi-fastidious bacteria, including some species of
Brucella, Corynebacterium, Listeria, Neisseria, and Vibrio.

Cetrimide agar
Cetrimide agar is a type of agar used for the selective isolation of the gram-negative
bacteria, Pseudomonas aeruginosa.

Tinsdale agar contains potassium tellurite, which can isolate Corynebacterium diphteriae
Fungal media
Bottom view of a Sabouraud agar plate with a colony of Trichophyton rubrum var. rodhaini.

Sabouraud agar
Sabouraud agar is used to culture fungi and has a low pH that inhibits the growth of most
bacteria; it also contains the antibiotic gentamicin to specifically inhibit the growth of
Gram-negative bacteria.

Hay infusion agar
Specific for the culturing of slime moulds (which are not fungi).

Potato dextrose agar
PDA is used to culture certain types of fungi.

Malt extract agar
Malt extract agar has a high content of peptone and is acidic. It is essentially used in the
isolation of fungal microorganisms.
Enriched media
Enriched media contain the nutrients required to support the growth of a wide variety of
organisms, including some of the more fastidious ones. They are commonly used to harvest as
many different types of microbes as are present in the specimen. Blood agar is an enriched
medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar
is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the
color for which it is named.
PART C:
1.Culture
Media for the Growth of Bacteria
For any bacterium to be propagated for any purpose it is necessary to provide the appropriate
biochemical and biophysical environment. The biochemical (nutritional) environment is made
available as a culture medium, and depending upon the special needs of particular bacteria (as
well as particular investigators) a large variety and types of culture media have been developed
with different purposes and uses. Culture media are employed in the isolation and maintenance
of pure cultures of bacteria and are also used for identification of bacteria according to their
biochemical and physiological properties.
The manner in which bacteria are cultivated, and the purpose of culture media, varies widely.
Liquid media are used for growth of pure batch cultures, while solidified media are used widely
for the isolation of pure cultures, for estimating viable bacterial populations, and a variety of
other purposes. The usual gelling agent for solid or semisolid medium is agar, a hydrocolloid
derived from red algae. Agar is used because of its unique physical properties (it melts at 100 oC
and remains liquid until cooled to 40oC, the temperature at which it gels) and because it cannot
be metabolized by most bacteria. Hence as a medium component it is relatively inert; it simply
holds (gels) nutrients that are in aquaeous solution.
Types of Culture Media
Culture media may be classified into several categories depending on their composition or use. A
chemically-defined (synthetic) medium (Table 4a and 4b) is one in which the exact chemical
composition is known. A complex (undefined) medium (Table 5a and 5b) is one in which the
exact chemical constitution of the medium is not known. Defined media are usually composed
of pure biochemicals off the shelf; complex media usually contain complex materials of
biological origin such as blood or milk or yeast extract or beef extract, the exact chemical
composition of which is obviously undetermined. A defined medium is a minimal medium
(Table 4a) if it provides only the exact nutrients (including any growth factors) needed by the
organism for growth. The use of defined minimal media requires the investigator to know the
exact nutritional requirements of the organisms in question. Chemically-defined media are of
value in studying the minimal nutritional requirements of microorganisms, for enrichment
cultures, and for a wide variety of physiological studies. Complex media usually provide the full
range of growth factors that may be required by an organism so they may be more handily used
to cultivate unknown bacteria or bacteria whose nutritional requirement are complex (i.e.,
organisms that require a lot of growth factors, known or unknown). Complex media are usually
used for cultivation of bacterial pathogens and other fastidious bacteria.
Figure 2. Legionella pneumophila. Direct fluorescent antibody (DFA) stain of a patient
respiratory tract specimen. © Gloria J. Delisle and Lewis Tomalty. Queens University,
Kingston, Ontario, Canada. Licensed for use by ASM Microbe Library
http://www.microbelibrary.org. In spite of its natural occurrence in water cooling towers
and air conditioners, Legionella is a fastidious bacterium grown in the laboratory, which
led to the long lag in identification of the first outbreak of Legionnaire's disease in
Philadelphia in 1977. Had fluorescent antibody to the bacterium been available at that
time, diagnosis could have been made as quickly as the time to prepare and view this slide.
Most pathogenic bacteria of animals, which have adapted themselves to growth in animal tissues,
require complex media for their growth. Blood, serum and tissue extracts are frequently added to
culture media for the cultivation of pathogens. Even so, for a few fastidious pathogens such as
Treponema pallidum, the agent of syphilis, and Mycobacterium leprae, the cause of leprosy,
artificial culture media and conditions have not been established. This fact thwarts the the ability
to do basic research on these pathogens and the diseases that they cause.
Other concepts employed in the construction of culture media are the principles of selection and
enrichment. A selective medium is one which has a component(s) added to it which will inhibit
or prevent the growth of certain types or species of bacteria and/or promote the growth of desired
species. One can also adjust the physical conditions of a culture medium, such as pH and
temperature, to render it selective for organisms that are able to grow under these certain
conditions.
A culture medium may also be a differential medium if allows the investigator to distinguish
between different types of bacteria based on some observable trait in their pattern of growth on
the medium. Thus a selective, differential medium for the isolation of Staphylococcus aureus,
the most common bacterial pathogen of humans, contains a very high concentration of salt
(which the staph will tolerate) that inhibits most other bacteria, mannitol as a source of
fermentable sugar, and a pH indicator dye. From clinical specimens, only staph will grow. S.
aureus is differentiated from S. epidermidis (a nonpathogenic component of the normal flora) on
the basis of its ability to ferment mannitol. Mannitol-fermenting colonies (S. aureus) produce
acid which reacts with the indicator dye forming a colored halo around the colonies; mannitol
non-fermenters (S. epidermidis) use other non-fermentative substrates in the medium for growth
and do not form a halo around their colonies.
An enrichment medium employs a slightly different twist. An enrichment medium (Table 5a
and 5b) contains some component that permits the growth of specific types or species of bacteria,
usually because they alone can utilize the component from their environment. However, an
enrichment medium may have selective features. An enrichment medium for nonsymbiotic
nitrogen-fixing bacteria omits a source of added nitrogen to the medium. The medium is
inoculated with a potential source of these bacteria (e.g. a soil sample) and incubated in the
atmosphere wherein the only source of nitrogen available is N2. A selective enrichment medium
(Table 5b) for growth of the extreme halophile (Halococcus) contains nearly 25 percent salt
[NaCl], which is required by the extreme halophile and which inhibits the growth of all other
procaryotes.
Table 4a. Minimal medium for the growth of Bacillus megaterium. An example of a
chemically-defined medium for growth of a heterotrophic bacterium.
Component Amount Function of component
sucrose
10.0 g
C and energy source
K2HPO4
2.5 g
pH buffer; P and K source
KH2PO4
2.5 g
pH buffer; P and K source
(NH4)2HPO4 1.0 g
pH buffer; N and P source
MgSO4 7H2O 0.20 g
S and Mg++ source
FeSO4 7H2O 0.01 g
Fe++ source
MnSO4 7H2O 0.007 g Mn++ Source
water
985 ml
pH 7.0
Table 4b. Defined medium (also an enrichment medium) for the growth of Thiobacillus
thiooxidans, a lithoautotrophic bacterium.
Component
Amount Function of component
NH4Cl
0.52 g
N source
KH2PO4
0.28 g
P and K source
MgSO4 7H2O
0.25 g
S and Mg++ source
CaCl2 2H2O
0.07 g
Ca++ source
Elemental Sulfur 1.56 g
Energy source
CO2
5%*
C source
water
1000 ml
pH 3.0
* Aerate medium intermittently with air containing 5% CO2.
Table 5a. Complex medium for the growth of fastidious bacteria.
Component Amount Function of component
Beef extract 1.5 g
Source of vitamins and other growth factors
Yeast extract 3.0 g
Source of vitamins and other growth factors
Peptone
6.0 g
Source of amino acids, N, S, and P
Glucose
1.0 g
C and energy source
Agar
15.0 g
Inert solidifying agent
water
1000 ml
pH 6.6
Table 5b. Selective enrichment medium for growth of extreme halophiles.
Component
Function
Amou of
nt
componen
t
Source of
amino
acids, N, S
and P
Casamino acids
7.5 g
Yeast extract
Source of
10.0 g growth
factors
Trisodium citrate
3.0 g
C
and
energy
source
KCl
2.0 g
K+ source
MgSO4 7 H2O
S
and
++
20.0 g Mg
source
FeCl2
0.023 Fe++
g
source
NaCl
Na+ source
for
halophiles
and
250 g
inhibitory
to
nonhalophi
les
water
1000
ml
pH 7.4
3.Tabulated differential media
MacCONKEY
AGAR EMB
AGAR
MacCONKEY AGAR
special
Bact.
102 Levine's
usual formulation
modification
formulation
selective
agent(s)
bile
crystal
neutral red
salts, bile
violet, crystal
neutral red
salts,
eosin
Y,
violet,
methylene blue
source
of
amino acids
which may peptone,
be
proteose peptone
deaminated
(alkaline rx.)
peptone,
proteose peptone
peptone
amino acid
added
for
detection of
none
decarboxylat
ion
(alkaline rx.)
none
none
fermentable
sugar(s)
(acid rx.)
pH indicator
lactose (1%)
lactose (1%)
lactose (1%)
neutral
red: neutral
net acid = red, net
acid
=
net
alkaline
= net
alkaline
whitish/light
whitish/light
eosin Y and
red: methylene
red, blue:
= net acid = dark,
net alkaline =
light
source from
which H2S
none
may
be
produced
Na thiosulfate
none
indicator of
H2S
none
production
ferric
citrate
none
HEKTOEN
AGAR
selective agent(s)
bile salts
ammonium
ENTERIC BRILLIANT
GREEN AGAR
brilliant green
XLD AGAR
Na desoxycholate
proteose
source of amino acids which may be proteose peptone,
peptone,
deaminated (alkaline rx.)
yeast extract
yeast extract
yeast extract
amino acid added for detection of
none
decarboxylation (alkaline rx.)
lysine
fermentable sugar(s) (acid rx.)
none
lactose
(1.2%),
lactose (0.75%),
lactose
(1%),
sucrose
(1.2%),
sucrose (0.75%),
sucrose (1%)
salicin (0.2%)
xylose (0.375%)
pH indicator
brom thymol blue +
acid
fuchsin:
net acid = yelloworange,
net alkaline = bluegreen
source from which H2S may be
Na thiosulfate
produced
indicator of H2S production
ferric
citrate
ammonium
phenol
red:
net
acid
= phenol
red:
yellow,
net acid = yellow,
net alkaline = net alkaline = red
red
none
Na thiosulfate
none
ferric ammonium
citrate
UNIT – III
Biochemical tests – identification of organisms - Susceptibility testing, reporting of results and
interpretation.
1. R factors:
A. Are small plasmids which encode resistance to only one type of antibiotic
B. Contain plasmid elements (replication origins, incompatibility determinants, etc.) that were
widespread in the pre-antibiotic era
C. Represent genetically engineered cloning vectors which have escaped into pathogenic bacteria
D. All of the above are correct
Correct Answer: B
2. Movement of DNA from one bacteria to another through a tubular bridge or pilus:
A. Conjugation
B. Transposition
C. Transfection
D. Transduction
Correct Answer: A
3. Which statement describing the potential advantages of DNA technologies over conventional
culture-based methods is not true?
A. Greater stability of samples during transport
B. Potentially more sensitive detection
C. More complete and accurate determination of organism resistance to antibiotics
D. More rapid than culture
Correct Answer: C
4. The polymerase chain reaction (PCR):
A. Has been adapted for accurate quantification of viruses
B. May yield false positive results when amplicons contaminate clinical samples
C. Offers detection sensitivity which often but not always exceeds that of culture
D. All of the above
Correct Answer: D
5. Which of the following is not one of Koch's postulates?
A. The organism is regularly found in lesions of the disease
B. The organism can be isolated from diseased tissues in pure culture on artificial media
C. Inoculation of this pure culture produces a similar disease in experimental animals
D. Treatment of the disease with a broad spectrum oral antimicrobial dependably eradicates the
organism and cures the disease
Correct Answer: D
PART B:
1.Biochemical tests?
Indole production test
A loop full of culture was inoculated in sterile Tryptone broth and then incubated at 37°C
for 48 hours. The formation of cherry red color in the top layer of the tube after the addition of
1ml of Kovac’s reagent indicates the positive reaction.
Methyl red test
A loop full of culture was inoculated in sterile MR-VP broth and then incubated at 35°C
for 48 hours. Formation of red color on adding 5 drops of methyl red indicator shows the positive
reaction.
Voges Proskauer test
A loop full of culture was inoculated in sterile MR-VP broth tubes and then incubated at
37°C for 24 - 48 hours. Following incubation two drops of  - Napthal solution and two drops of
40% KOH were added. The development of crimson to ruby pink (red) color indicates positive
reaction.
Citrate utilization test
A loop full of culture was streaked in sterile Simmon’s citrate agar slants and incubated
at 37°C for 48 hours. Bromo phenol blue was used as a pH indicator. Change of color from green
to Prussian blue indicates the positive reaction
Oxidase test
A loop full of culture was inoculated in Trypticase say agar and then incubated at 37°C
for 24 hours.
The oxidase disc impregnated with Tetra Methyl Para Phenylene Diamine
Dihydrochloride was placed on the surface of the colony. Appearance of deep blue colour within
10 seconds indicates positive reaction.
Catalase test
A loop full of culture was inoculated in Nutrient agar slants. The tubes were incubated at
37°C for 24 hours. Then 1ml of H2O2 was added to the surface of the slants. Appearance of air
bubbles indicates positive reaction.
Urease test
A loopful of culture was inoculated in sterile Christenson’s urea agar slants and incubated
at 35°C for 24 hours. A deep pink color formation at the end of incubation indicates the positive
reaction.
Nitrate reduction test
A loop full of culture was inoculated in sterile Nitrate broth tubes and then incubated at
37°C for 96 hours. 0.1mL of test reagent (5g of  - Napthal and 8g of sulphanilic acid in acetic
acid) was added. A red colour development indicates the positive reaction.
Triple sugar Iron agar
A loop full of culture was streaked in sterile Triple sugar Iron agar slants and incubated at
37°C for 24 hours. Blackening of the test medium was recorded as positive for the Hydrogen
Sulphide production. An alkaline reaction was indicated by a red coloration and the production
of acid by yellowing of the medium. Air bubbles indicate gas production.
Carbohydrate fermentation test
A loop full of culture was inoculated in sterile peptone broth incorporated with 1% of
different sugars such as glucose, maltose, mannitol, sucrose and lactose and incubated at 37°C
for 24 – 48 hours. Change of broth color green to yellow indicated acid production and the
presence of air bubble in the Durham’s tube indicates the gas production.
.
Hydrogen sulphide production test
A loop full of culture was stabbed in SIM agar tubes (Sulfide Indole Motility agar). The
tubes were incubated at 37°C for 48 hours. Black coloration along the line of stab inoculation
indicates the positive reaction for H2S production.
Starch hydrolysis
A loop full of culture was taken and a single line streak inoculation was made in sterile
starch agar medium plates. It was then incubated at 37°C for 48 hours. The plates were then
flooded with iodine solution. A clear zone surrounding the microbial growth indicates the
positive reaction.
Gelatin hydrolysis
A loop full of culture was stabbed in sterile Gelatin agar tubes. It was then incubated at
37°C for 24 hours. After incubation the tubes were placed in refrigerator at 4°C for 30 minutes.
Liquefaction of gelatin medium at 4°C indicates the positive reaction.
PART C:
1. SUSCEPTIBILITY TESTING
In Vitro Antibiotic Susceptibility Testing
Performed to determine the susceptibility of the organism isolated from the diseased
host; Generate antibiogram(s)
Broth Dilution MIC test
Agar Dilution MIC test
Broth/Agar MBC test
Agar Diffusion (Kirby-Bauer Disk) Test: Measure diameter of zone of inhibition; Read
as susceptible, intermediate, or resistant
Factors influencing zones of inhibition on agar
Concentration of
Pathogen
Antibiotic
Agar
Growth
Temperature
Nutrient
Drug antagonists
bacteria
spread
diffusion
onto
agar plate
susceptibility
effects
depth
rate
availability
Factors influencing diffusion of antibiotic
Concentration of antibiotic: Kirby-Bauer disks, E-tests have
standardized
concentrations
Molecular
weight
of
antibiotic
Water
solubility
of
antibiotic
pH
and
ionization
Binding to agar
Modern Commercial Kits (e.g., E-test strips (up to six) on an agar plate and can be
read out as MIC)
Minimal Inhibitory Concentration (MIC)
(Broth Tube Dilution Method)
The tube dilution test is the standard method for determining levels of resistance to
an antibiotic. Serial dilutions of the antibiotic are made in a liquid medium which is
inoculated with a standardized number of organisms and incubated for a prescribed
time. The lowest concentration (highest dilution) of antibiotic preventing appearance
of turbidity is considered to be the minimal inhibitory concentration (MIC). At this
dilution the antibiotic is bacteriostatic.
Additionally, the minimal bactericidal concentration (MBC) can be determined by
subculturing the contents of the tubes onto antibiotic-free solid medium and
examining for bacterial growth.
Although the tube dilution test is fairly precise, the test is laborious because serial
dilutions of the antibiotic must be made and only one isolate can be tested in each
series of dilutions.
Procedure:
1. Number sterile capped test tubes 1 through 9. All of the
following steps are carried out using aseptic technique.
2. Add 2.0 ml of tetracycline solution (100 ug/ml) to the first
tube. 3. Add 1.0 ml of sterile broth to all other tubes.
3. Transfer 1.0 ml from the first tube to the second tube.
4. Using a separate pipette, mix the contents of this tube and
transfer 1.0 ml to the third tube.
5. Continue dilutions in this manner to tube number 8, being
certain to change pipettes between tubes to prevent
carryover of antibiotic on the external surface of the
pipette.
6. Remove 1.0 ml from tube 8 and discard it. The ninth tube,
which serves as a control, receives no tetracycline.
7. Suspend to an appropriate turbidity several colonies of the
culture to be tested in 5.0 ml of Mueller-Hinton broth to
give a slightly turbid suspension.
8. Dilute this suspension by aseptically pipetting 0.2 ml of the
suspension into 40 ml of Mueller-Hinton broth.
9. Add 1.0 ml of the diluted culture suspension to each of the
tubes. The final concentration of tetracycline is now onehalf of the original concentration in each tube.
10. Incubate all tubes at 35oC overnight.
11. Examine tubes for visible signs of bacterial growth. The
highest dilution without growth is the minimal inhibitory
concentration (MIC
Minimal Bactericidal Concentration (MBC)
The Minimal Bactericidal Concentration (MBC) assay is performed as an adjunct to
the MIC and is used to determined the concentration of antibiotic that is lethal to the
target bacteria in vitro.
Procedure:
1. From each MIC broth tube without visible growth,
aliquot a 100 ml volume of the broth onto MuellerHinton agar and spread across the entire surface of
the plate.
2. Record the dilution of the subcultured MIC tube on
each plate and incubate at 35oC until the next lab
session.
3. Following overnight incubation, examine the MBC
plates for colony growth or lack of growth for each
dilution subcultured. No growth indicates that the
antibiotic was bactericidal at that dilution. Growth
indicates that the antibiotic was bacteriostatic but not
bactericidal at that dilution.
---------------------------------------------------------------------------------------------------------------------------------
Antibiotic Disk Susceptibilities
(Kirby-Bauer Disk-Diffusion Method)
The disk-diffusion method (Kirby-Bauer) is more suitable for routine testing in a
clinical laboratory where a large number of isolates are tested for susceptibility to
numerous antibiotics. An agar plate is uniformly inoculated with the test organism and
a paper disk impregnated with a fixed concentration of an antibiotic is placed on the
agar surface. Growth of the organism and diffusion of the antibiotic commence
simultaneously resulting in a circular zone of inhibition in which the amount of
antibiotic exceeds inhibitory concentrations. The diameter of the inhibition zone is a
function of the amount of drug in the disk and susceptibility of the microorganism.
This test must be rigorously standardized since zone size is also dependent on
inoculum size, medium composition, temperature of incubation, excess moisture and
thickness of the agar. If these conditions are uniform, reproducible tests can be
obtained and zone diameter is only a function of the susceptibility of the test
organism.
Zone diameter can be correlated with susceptibility as measured by the dilution
method. Further correlations using zone diameter allow the designation of an
organism as "susceptible", "intermediate", or "resistant" to concentrations of an
antibiotic which can be attained in the blood or other body fluids of patients requiring
chemotherapy.
Procedure:
1. Make a suspension at an appropriate turbidity of the bacterial
culture to be tested.
2. Place a sterile cotton swab in the bacterial suspension and remove
the excess fluid by pressing and rotating the cotton against the
inside of the tube above the fluid level. The swab is streaked in at
least three directions over the surface of the Mueller-Hinton agar
to obtain uniform growth. A final sweep is made around the rim
of the agar. Be sure to streak for confluency.
3. Allow the plates to dry for five minutes.
4. Using sterile forceps, place disks containing the following
antibiotics on the plate: penicillin G, ampicillin, cephalothin,
erythromycin, tetracycline, methicillin, streptomycin or other
appropriate antibiotic disks.
5. Incubate the plates within 15 minutes after applying the disks. The
plates should be incubated soon after placing the disks since the
test is standardized under conditions where diffusion of the
antibiotic and bacterial growth commence at approximately the
same time.
6. Following overnight incubation, measure the diameter of the zone
of growth inhibition around each disk to the nearest whole mm.
Examine the plates carefully for well-developed colonies within
the zone of inhibition.
7. Using a standard table of antibiotic susceptibilities, determine if
the strain is resistant, intermediate, or susceptible to the antibiotics
tested.
How are the results reported?
Typically, the raw data are interpreted based on the available CLSI data. The results will
be reported out as:
Susceptible
'The "susceptible" category implies that isolates are inhibited by the usually achievable
concentrations of antimicrobial agent when the recommended dosage is used for the site
of infection.' (CLSI definition)
Note that this definition says nothing about the chances of clinical success; in fact
predicting clinical outcome based on susceptibility testing and the use of drugs shown to
be in the susceptible category is very imprecise. This imprecision is due to the effect of
host responses, site of infection, toxin production by bacteria that is independent of
antimicrobial susceptibility, the presence of biofilm, drug pharmacodynamics and other
factors.
Resistant
'The "resistant" category implies that isolates are not inhibited by the usually achieveable
concentrations of the agent with normal dosage schedules, and/or that demonstrate zone
diameters that fall in the range where specific microbial resistance mechanisms (e.g. betalactamases) are likely, and clinical efficacy of the agent against the isolate has not been
reliably shown in treatment studies.' (CLSI definition).
Note that this definition says nothing about the chances of clinical success; in fact
predicting clinical outcome based on susceptibility testing and the use of drugs shown to
be in the resistant category is imprecise. This imprecision is due to the effect of host
responses, site of infection, toxin production by bacteria that is independent of
antimicrobial susceptibility, the presence or absence of biofilm, drug pharmacodynamics
and other factors. However, with the exception of urinary bladder infections and some
mycobacterial infections, most clinicians avoid the use of a "resistant" category drug to
treat infection.
Intermediate
'The "intermediate" category includes isolates with antimicrobial MICs that approach
usually attainable blood and tissue levels and for which response rates may be lower than
for susceptible isolates. The intermediate category implies clinical efficacy in body sites
where the drugs are physiologically concentrated (e.g. quinolones and beta-lactams in
urine) or when a higher than normal dosage of a drug can be used (e.g. betalactams). This
category also includes a buffer zone, which should prevent small, uncontrolled, technical
factors from causing major discrepancies in interpretations, especially for drugs with
narrow pharamacotoxicity margins.' (CLSI definition)
UNIT – IV
Serology – Antigen - antibody reactions – Agglutinations (blood grouping, WIDAL),
Precipitation (VDRL), Immunodiffusion – mono and double immunodiffusion,
Immunoelectorophoresis (rocket, counter current).
PART A:
1. The diagnosis of septicemia should be considered in patients who are at increased risk of
blood stream infection (often secondary to local disease, such as urinary tract infection or
pneumonia). All of the following are factors predisposing patients to septicemia except:
A. Underlying diseases that appear to compromise host defenses, such as diabetes, lymphoma,
etc.
B. Patients with a polymorphonuclear leukocyte count less than 1000/mm3
C. Patients with polymorphonuclear leukocyte counts of 10,000 to 20,000
D. Long term therapy with broad-spectrum antimicrobials.
Correct Answer: C
2. Physical findings and other factors that should suggest the presence of septicemia in a
particular patient are all of the following except:
A. Shaking chills, spiking fevers
B. Conjunctivitis
C. Nausea, vomiting, diarrhea
D. In elderly patients, decreased urine output or mental changes (confusion)
Correct Answer: B
3. The most likely organism to be causing septicemia depends, among other things, on the
patients' personal risk factors (e.g., their underlying disease), or lab data (e.g. their leukocyte
count), "community risk factors" (such as what particular organisms inhabit the unit on which
they happen to be in the hospital); and the physical examination. In a bone marrow transplant
patient with a fever of 104 F, who has a white blood cell count of 345/mm3, there are scattered
skin lesions comprised of a central blue area with an areola of redness (inflammation). You
vaguely remember seeing such a lesion before in a lecture. "Ecthyma something or the other" is
the best you can do..."Oh yes, with the blue, gangrene-like center, ecthyma gangrenosum, that's
it" is your mental conversation. In this patient the most likely organism is:
A. Reimerbacterium deaniium
B. Pseudomonas aeruginosa
C. Flavobacterium aniseum
D. Godzillobacterium resistium
Correct Answer: B
4. It is extremely difficult to eradicate staphylococcal infection in the presence of a foreign body.
Thus, infected artificial joints and infected intravenous lines often must be removed in
conjunction with optimal intravenous anti-staphylococcal therapy with oxacillin or nafcillin
(sometimes in combination with rifampin) in order to eradicate such staphylococcal infections
(and these attempts are not always successful). Staphylococcal infection of bone may persist for
many years despite what should be optimal antimicrobial therapy. The most likely reason for this
tenacity of staphylococcal osteomyelitis is:
A. The Staphylococcal pilus gene is up-regulated by oxacillin
B. Fragments of dead bone, called "involucrum" may act as a foreign body, and may have to be
removed for optimal eradication of the staphylococcal infection.
C. Staphylococci may be perceived by the patient's immune system as a positive factor, because
the staphylococci produce an extracellular enzyme, obtundokinase, which makes the patient
more alert.
D. Staphylococci are such an integral part of our normal flora that they are treated as "self" rather
than "non-self", and no immune reaction to them occurs.
Correct Answer: B
5. A 42 year old woman who was complaining of shaking chills and fever went to the Salt Lake
City Homeless clinic. She had enlarged lymph nodes in the right axilla. "Upstream" from the
enlarged nodes was an ulcer on the top of the patient's right hand. When carefully questioned, the
patient said she remembered being bitten there by an insect resembling a horsefly, but with
yellow stripes on its abdomen; "Its what we used to call a deerfly," she added laconically. The
patient denied any contact with rats or fleas. The patient's disease was most likely to be:
A. Brucellosis
B. Ulcero-glandular tularemia
C. Pneumonic plague
D. Relapsing fever
Correct Answer: B
PART B:
1. Explain about general characteristics of the antibody response.
I. GENERAL CHARACTERISTICS OF THE ANTIBODY RESPONSE
A.
Self/non-self
discrimination
One characteristic feature of the specific immune system is that it normally distinguishes between
self and non-self and only reacts against non-self.
B.
Memory
A second feature of the specific immune response is that it demonstrates memory. The immune
system "remembers" if it has seen an antigen before and it reacts to secondary exposures to an
antigen in a manner different than after a primary exposure. Generally only an exposure to the same
antigen will illicit this memory response.
C.
Specificity
A third characteristic feature of the specific immune system is that there is a high degree of
specificity in its reactions. A response to a particular antigen is specific for that antigen or a few
closely related antigens.
N.B. These are characteristic of all specific immune responses.
II. ANTIBODY FORMATION
A. Fate of the immunogen
1. Clearance after primary injection - The kinetics of antigen clearance from the body after a
primary administration is depicted in Figure 1.
a) Equilibrium phase - The first phase is called the equilibrium or equilibration phase. During this
time the antigen equilibrates between the vascular and extravascular compartments by diffusion.
This is normally a rapid process. Since particulate antigens don't diffuse, they do not show this
phase.
b) Catabolic decay phase - In this phase the host's cells and enzymes metabolize the antigen. Most of
the antigen is taken up by macrophages and other phagocytic cells. The duration will depend upon
the immunogen and the host.
c) Immune elimination phase - In this phase newly synthesized antibody combines with the antigen
producing antigen/antibody complexes which are phagocytosed and degraded. Antibody appears in
the serum only after the immune elimination phase is over.
2. Clearance after secondary injection - If there is circulating antibody in the serum injection of
the antigen for a second time results in a rapid immune elimination. If the is no circulating antibody
then injection of the antigen for a second time results in all three phases but the onset of the immune
elimination phase is accelerated.
B. Kinetics of antibody responses to T-dependent Antigen
1. Primary (1o) Antibody response - The kinetics of a primary antibody response to and antigen is
illustrated in Figure 2.
a) Inductive, latent or lag phase - In this phase the antigen is recognized as foreign and the cells
begin to proliferate and differentiate in response to the antigen. The duration of this phase will vary
depending on the antigen but it is usually 5-7 days.
b) Log or Exponential Phase - In this phase the antibody concentration increases exponentially as
the B cells that were stimulated by the antigen differentiate into plasma cells which secrete antibody.
c) Plateau or steady-state phase - In this phase antibody synthesis is balanced by antibody decay so
that there in no net increase in antibody concentration.
d) Decline or decay phase - In this phase the rate of antibody degradation exceeds that of antibody
synthesis and the level of antibody falls. Eventually the level of antibody may reach base line
levels.
2. Secondary (2o), memory or anamnestic response (Figure 3)
a) Lag phase - In a secondary response there is a lag phase by it is normally shorter than that
observed in a primary response.
b) Log phase - The log phase in a secondary response is more rapid and higher antibody levels are
achieved.
c) Steady state phase
d) Decline phase - The decline phase is not as rapid and antibody may persist for months, years or
even a lifetime.
C. Specificity of 1o and 2o responses
Antibody elicited in response to an antigen is specific for that antigen although it may also cross
react with other antigens which are structurally similar to the eliciting antigen. In general secondary
responses are only elicited by the same antigen used in the primary response. However, in some
instances a closely related antigen may produce a secondary response, but this is a rare exception.
D. Qualitative changes in antibody during 1o and 2o responses
1. Ig class variation - In the primary response the major class of antibody produced is IgM whereas
in the secondary response it is IgG (or IgA or IgE) (Figure 4). The antibodies that persist in the
secondary response are the IgG antibodies.
2. Affinity - The affinity of the IgG Ab produced increases progressively during the response,
particularly after low doses of antigen (Figure 5). This is referred to as affinity maturation. Affinity
maturation is most pronounced after secondary challenge with antigen.
One explanation for affinity maturation is clonal selection as illustrated in Figure 6. A second
explanation for affinity maturation is that, after a class switch has occurred in the immune response,
somatic mutations occur which fine tune the antibodies to be of higher affinity. There is
experimental evidence for this mechanism, although it is not known how the somatic mutation
mechanism is activated after exposure to antigen.
3. Avidity - As a consequence of increased affinity, the avidity of the antibodies increases during
the response.
4. Cross-reactivity - As a result of the higher affinity later in the response there is also an increase
in detectible cross reactivity. An explanation for why increasing affinity results in an increase in
detectible cross reactivity is illustrated by the following example.
Affinity of Ab for Ag
Immunizing Ag
Cross reacting Ag
Early
Late
10-6
10-9
+
++
10-3
10-6
-
+
If a minimum affinity of 10-6 is needed to detect a reaction, early in an immune response the reaction
of a cross reacting antigen with an affinity of 10-3 will not be detected. However, late in a response
when the affinities increase 1000 fold, the reaction with both the immunizing and cross reacting
antigens will be detected.
E. Cellular events during 1o and 2o responses to T-dependent antigen
1. Primary response (Figure 7)
a) Lag phase - Clones of T and B cells with the appropriate antigen receptors bind antigen, become
activated and begin to proliferate. The expanded clones of B cells differentiate into plasma cells
which begin to secrete antibody.
b) Log phase - The plasma cells initially secrete IgM antibody since the Cμ heavy chain gene is
closest to the rearranged VDJ gene. Eventually some B cells switch from making IgM to IgG, IgA
or IgE. As more B cells proliferate and differentiate into antibody secreting cells the antibody
concentration increases exponentially.
c) Stationary phase - As antigen is depleted, T and B cells are no longer activated. In addition,
mechanisms which down regulate the immune response come into play. Furthermore, plasma cells
begin to die. When the rate of antibody synthesis equals the rate of antibody decay the stationary
phase is reached.
d) Decline phase - When no new antibody is produced because the antigen is no longer present to
activate T and B cells and the residual antibody slowly is degraded, the decay phase is reached.
2. Secondary response (Figure 8)
Not all of the T and B cells that are stimulated by antigen during primary challenge with antigen die.
Some of them are long lived cells and constitute what is refer to as the memory cell pool. Both
memory T cells and memory B cells are produced and memory T cells survive longer than memory
B cells. Upon secondary challenge with antigen not only are virgin T and B cells activated, the
memory cells are also activated and thus there is a shorter lag time in the secondary response. Since
there is an expanded clone of cells being stimulated the rate of antibody production is also increased
during the log phase of antibody production and higher levels are achieved. Also, since many if not
all of the memory B cells will have switched to IgG (IgA or IgE) production, IgG is produced earlier
in a secondary response. Furthermore since there is an expanded clone of memory T cells which can
help B cells to switch to IgG (IgA or IgE) production, the predominant class of Ig produced after
secondary challenge is IgG (IgA or IgE).
F. Ab response to T-independent antigen - Responses to T-independent antigen are characterized
by the production of almost exclusively IgM antibody and no secondary response. Secondary
exposure to the antigen results in another primary response to the antigen as illustrated in Figure 9.
G. Class switching
During an antibody response to a T-dependent antigen a switch occurs in the class of Ig produced
from IgM to some other class (except IgD). Our understanding of the structure of the
immunoglobulin genes, helps explain how class switching occurs (Figure 10).
During class switching another DNA rearrangement occurs between a switch site (S μ) in the intron
between the rearranged VDJ regions and the Cμ gene and another switch site before one of the other
heavy chain constant region genes. The result of this recombination event is to bring the VDJ region
close to one of the other constant region genes, thereby allowing expression of a new class of heavy
chain. Since the same VDJ gene is brought near to a different C gene and since the antibody
specificity is determined by the hypervariable regions within the V region, the antibody produced
after the switch occurs will have the same specificity as before.
Cytokines secreted by T helper cells can cause the switch to certain isotypes.
H. Membrane and secreted immunoglobulin
The specificity of membrane immunoglobulin on a B cell and the Ig secreted by the plasma cell
progeny of a B cell is the same. An understanding of how the specificity of membrane and secreted
Ig from an individual B cell can be the same comes from an understanding of immunoglobulin
genes (Figure 11).
There are two potential polyA sites in the immunoglobulin gene. One after the exon for the last
heavy chain domain and the other after the exons that code for the trans- membrane domains. If the
first polyA site is used, the pre-mRNA is processed to produce a secreted protein. If the second
polyA site is used, the pre-mRNA is processed to produce a membrane form of the immunoglobulin.
However, in all cases the same VDJ region is used and thus the specificity of the antibody remains
the same. All C regions genes have these additional membrane pieces associated with them and thus
after class switching other classes of immunoglobulins can be secreted or expressed on the surface
of B cells.
Serology
Serotyping
Precipitation
Agglutination
Complement Activation
Cross-linking
Particulate antigens
Titer
Don’t worry too much about the theoretics of the Primary Interactions between Antibody and
Antigen except for this:
The word affinity is used to describe the strength of binding between one antibody
binding site and an antigenic determinant (epitope or hapten). The association constant
is the mathematical value that is the measure of the strength of binding.
Anibody molecules are multivalent and antigens are also often multivalent. This
multivalency tends to increase the strength of the interaction, and this really represents
the true state of affairs. This overall binding energy that results in the binding of a
multivalent antibody with a multivalent antigen is called the functional affinity or the
avidity.
PART C:
1. Write a note on Agglutination reaction.
Agglutination Reactions
The discussion covering zeta Potential and the Coombs Test (which is also known as the antiglobulin test or the anti-immunoglobulin test) is very important. What is the distinction
between the direct Coombs test and the indirect Coombs test?
Passive agglutination tests involve attaching an antigen to a particle of some sort (latex beads,
tanned red blood cells) and then running an agglutination reaction with antibody.
3.Write a note on Precipitation Reactions.
This describes the reaction between soluble antibody and soluble antigen in which an insoluble
product results. Please note the discussion describing the effects of antibody excess, antigen
excess, and the zone of optimal proportions (equivalence zone) on the production of a
precipitate. (Remember that precipitation is a secondary phenomenon. Ag-Ab reactions may
occur and form soluble immune complexes even without the production of a visible
precipitate!)
Precipitation reactions can be done in a variety of ways:
In test tubes
In agarose gels:
As double diffusion
As single diffusion
After electrophoresis
Immunoassays
Solid phase immunoassays;
ELISA – Enzyme linked immunosorbant assay
1. 1. You need reagent antibodies or reagent binding proteins that have been
“tagged” with an enzyme label. This means that the enzyme has been
covalently coupled to the protein reagent.
a. a.
Typical enzymes include: Horseradish peroxidase or Alkaline
phosphatase
b. b. Reagent proteins include antibodies such as goat-anti-human-IgG.
Or, bacterial proteins that bind to antibodies such as Staphylococcal
protein A or Streptococcal protein G. Biotin and Avidin can also be
used ---- avidin has several high-affinity binding sites for biotin thus it
can be used to bridge molecules that have been “tagged” with biotin.
2. 2. You need some kind of “solid phase” to which proteins can stick. This
would usually be some kind of plastic microtiter plate. Thus, as is the
example in Fig 5.11, antigen can be used to “naturally” coat the wells of a
microtiter plate. After you do this you would have to “block” the plate with
some kind of irrelevant protein (or detergent). We usually use a milk solution
for this that is called BLOTTO.
3. 3. One then reacts the antigen-coated plate with appropriate enzyme-tagged
antibody. (You can actually sandwich several different reagents at this point –
as will be discussed in class.) Afterwards the appropriate substrate is added
and a color-change will indicate a “positive” test.
Immunofluorescence
In these assays, antibodies or other reagent proteins are “tagged” or labeled with
fluorescent dyes. These fluorescent reagents can then be used to stain samples mounted
onto microscope slides and the slides can be examined using a fluorescent microscope.
Typical fluorescent labels include FITC, TRITC, PE and many others.
Cells in suspension can also be stained fluorescently and then analyzed by fluorescenceactivated cell sorting or FACS analyis.
Reagent Antibodies
For many of the serological tests described above, it is essential to have antibodies of
defined specificity which can be used as reagents in the tests.
Traditionally such antibodies were made as polyclonal antibodies. To make such
antibodies, very pure antigen was injected into subject animals and then after an
appropriate amount of time (often after several “booster shots”) the antibody containing
serum was harvested from the animal by bleeding.
Such polyclonal antisera
Particularly, such antisera
epitopes of the antigen
contaminated the antigen.
isotypes of antibody too.
are very valuable, but they do have some limitations.
contain populations of antibodies that react to all the
prep – including any impurities that might have
Of course, polyclonal antisera also contain multiple
To get around this problem, one can immunize an animal and then remove and culture
and clone its B-cells. In this technique, one then cultures single clones of B-cells, each
clone producing only one specificity of antibody, reactive with only one epitope of the
immunizing antigen.
This procedure involves a number of sophisticated tricks that we will list here and
discuss in more detail in class.
1. 1.
Normal B-cells will not survive very long in culture so one must
immortalize them by giving them genes from cancerous B-cells.

Cancerous plasma cells (antibody secreting B-cells) are called
myeloma cells.

B-cells from immunized animals are mixed with myeloma cells
in the presence of a mild detergent (polyethylene glycol or PEG).
This causes cells to fuse forming hybridomas or hybrids between
normal and myeloma cells.
2. 2. A selection system is used that preferentially allows the hybridoma cells
to grow while suppressing the growth of any non-hybridized myeloma cells.
3. 3. Hybridoma cells are fragile, so feeder cells of macrophages are used to
produce growth promoting cytokines.
4. 4. Appropriate immunoassays (usually ELISA’s) have that allow you detect
any antibodies being produced by the hybridoma clones.
3.Introduction to Techniques in Immunology
Introduction to the Immune System
Immunology emerged from medical science and has permeated all biology. The spread of
immunology into other fields has been the result of the scientist and clinician using immunologic
techniques as a sensitive analytical tool. Immunological assays are important in regulatory work
because of their sensitivity, specificity, and rapidity. To aid in understanding the theory behind
immunoassays a brief introduction to the immune system will be presented.
We live in a hostile world filled with many infectious agents; however, vertebrate animals
possess an effective immune system which prevents the invasions of parasites such as bacteria,
viruses, and cancer cells. The immune system specifically recognizes and selectively eliminates
foreign invaders. The immune systems responds in a specific way to pathogens and displays a
long-term
memory
of
earlier
contacts
with
the
disease
agents.
Vertebrates are protected by a dual immune system known as cell-mediated immunity and
humoral immunity. The two immune systems together provide an excellent defense against
foreign invaders. Both systems are adaptive and respond specifically to most foreign substances,
although depending on the antigen one immune response generally is favored over the other.
Cell-mediated immunity is particularly effective against fungi, parasites, intracellular viral
infections, cancer cells, and foreign tissue. The humoral immune response defends primarily
against extracellular bacteria and viral infections. The cells involved in both immune systems are
lymphocytes which originate in the bone marrow and migrate to different lymphoid organs.
There are two types of lymphocytes which are known as T cells and B cells. The two types of
lymphocytes are responsible for a dual immunity phases. All lymphocytes are derived from bone
marrow but those that pass through the thymus become T cells (T lymphocytes). The T cells are
responsible for cell-mediated immunity. Since the immunity involving T cells is associated with
the T cells themselves, this type of immunity is called cell-mediated immunity.
Other lymphocytes pass through the bursa of Fabricius in birds or the bursa equivalent in
mammals and become B cells (B lymphocytes). The bursa of Fabricius is the primary lymphoid
organ associated with the cloaca in birds but is not found in mammals. The bursa-derived
lymphocytes (B cells) produce antibodies which can react specifically with antigen. Because B
cells produce antibodies that circulate and the immunity is called humoral immunity. Antigen
specific antibodies produced in the humoral immune response can be used in immunological
assays to detect various disease agents or antigenic molecules. With the development of
monoclonal antibodies, greater specificity and reproducibility can be obtained with
immunological assays. The remainder of this discussion will be devoted to the humoral immune
response.
4.The Humoral Immune Response
Basic to the humoral immune response is the formation of antibodies (protein molecules)
generated in response to the presence of foreign substances. The foreign substances that induce
an immune response and interact with antibodies are called antigens (or immunogens). Antigens
are traditionally defined as any substance that, when introduced parenterally into an animal, will
cause the production of antibodies and will react specifically with the antibodies in vitro (Figure
2). Antigens are macromolecules (10,000 MW) that possess a high degree of internal chemical
complexity. They are soluble in water and foreign to the animal in which they stimulate
antibodies. We generally do not produce antibodies against our own body's molecules or against
low-molecular-weight
molecules
(less
than
10,000).
Haptens are low molecular weight molecules that are non-antigenic and cannot stimulate
antibody production by themselves; however, they will react with appropriate antibody
molecules. When haptens combine with a larger carrier molecule, they convey a new antigenic
determinant site to the carrier molecule. Drugs and pesticides are low molecular weight
molecules and can be treated as haptens. By conjugation to larger carrier molecules (albumin),
low molecular weight drugs and pesticides can be made antigenic. Antibodies produced in this
matter will react specifically with low molecular weight haptens.
Polysaccharides like heparin (17,000 mol wt) are also nonantigenic and do not produce
antibodies when injected into animals. The greater the molecular weight of a substance, the more
likely it is to function as an antigen. Within each molecule there are specific regions of limited
size (10,000 mol wt) that function as the antigenic determinant sites. Larger antigens will have
more antigenic determinant sites which means that many different antibodies will be produced in
response
to
a
large
antigen.
Molecular complexity High molecular weight is not enough to confer antigenicity on a foreign
substance. There must be internal complexity. The organic chemist can produce synthetic
polymers of any size but most are not antigenic unless there is internal complexity. Most
naturally occurring macromolecules are often very complex because they are built from many
different low molecular weight constituents. Proteins are very antigenic since they consist of 20
different
amino
acids.
Solubility Another argument used to explain the nonantigenicity of synthetic polymers is their
insolubility in body fluids. Many of the particulate antigens, bacteria and viruses are engulfed by
macrophages
and
digested
into
soluble
components.
Foreignness Antigens must be foreign to the host. The more foreign the antigen is to the host, the
better it will stimulate antibody formation. Duck serum proteins are not good antigens for
chickens, but antigen from bacteria are extremely antigenic and will stimulate the formation of
antibodies in chickens. Animals do not generally produce antibodies against self protein.
When a vertebrate first encounters an antigen, it exhibits a primary humoral immune response. If
the animal encounters the same antigen after a few days the immune resonse is more rapid and
has a greater magnitude (Figure 6). The initial encounter causes specific B-cell clones to
proliferate and differentiate. The progeny lymphocytes include not only effector cells (antibody
producing cells) but also clones of memory cells, which retain the capacity to produce both
effector and memory cells upon subsequent stimulation by the original antigen. The effector cells
live for only a few days; therefore, the antibody titer increases and decreases within 20 days. The
memory cells live for a lifetime and can be reactivated by a second stimuation with the same
antigen. Thus when an antigen is encountered a second time, its memory cells quickly produce
effector cells which rapidly produce massive quantities of antibodies. The secondary immune
response
is
also
called
the
anamnestic
response
or
booster
response.
Antibody are Y-shaped proteins found in sera which are produced in response to a specific
antigen. Antibodies have different molecular weights and sedimentation coefficients depending
on the class of antibodies. IgG has a molecular weight of 150,000 and a sedimentation
coefficient of 7S. The largest antibody molecule is IgM with a molecular weight of 900,000 and
a sedimentation coefficient of 19S. Antibodies are composed of two heavy peptide chains and
two short peptide chains (Figure 7). Two identical heavy chains have a molecular weight of
50,000 each, and two identical light chains have a molecular weight of 25,000 each. These
chains are connected by inter-disulfide bonds. Purified preparations of IgG are resistant to
reductive cleavage by sulfhydryl reagents unless the molecule was unfolded by high
concentrations of urea or guanidine. The number and precise position of both inter and intra
disulfide bonds differ and are a characteristic of the subclasses. At the amino terminal end of the
antibody is a short segment called the variable region. The amino acid sequence of the variable
region is different for each antibody and is specific for a certain antigen. Within each variable
region there is a hypervariable region. The hypervariable region of the antibody binds
specifically to the antigen in a lock-and-key manner. It is the variable region of the antibody
which
allows
development
of
sensitive
and
specific
immunoassays.
The carboxyl end of the heavy and light chain of the antibody molecule is called the constant
region. The amino acid sequence of the constant region is similar to the sequence of antibody in
the same class. The constant region is the part of the antibody that binds to mast cells,
complement, and protein A. Protein A is a bacterial cell wall protein isolated from
Staphylococcus aureus which binds to the Fc (Fragment-crystallizable) fragment of the antibody
molecule. This protein can be used in affinity chromatography to purify antibodies.
Papain digestion cleaves IgG molecules into two fragments which can be separated by
carboxymethyl cellulose ion exchange chromatography. One fragment will crystallize
spontaneously. This fragment is called Fc fragment (fragment-crystallizable) and is deficient in
antigen-binding ability. The Fc fragment has a molecular weight of approximately 50,000 and a
sedimentation coefficient of 3.5S. The Fc fragment is the constant region of the heavy chain with
a COOH terminus end. The second fragment is fragment antigen-binding (Fab). The molecular
weight and sedimentation coefficient for the Fab are similar to Fc fragments. An IgG molecule
consists of 1 Fc and 2 Fab fragments. Each Fab fragment consists of the amino terminal half of
the heavy and light chains. It was possible to predict the structure of IgG molecule with Papain
(Fc and Fab) fragments and sulfhydryl reagents which split the IgG molecule into light and
heavy
chains.
Antibodies specifically combine with a small segment of the antigen called the antigenic
determinant or epitope to form an antigen-antibody complex. The antigen-antibody reaction is
characterized by specificity. If a mouse is injected with goat albumin, the mouse will produce
antigoat albumin antibodies. These specific antibodies will react only with goat albumin or
albumin that is closely related. This process is referred to as a polyclonal response. The
polyclonal response is partly due to the different clones of B cells of the immune system and
partly due to the complex nature of antigens. The polyclonal response suggests that different
clones of B cells are producing five different classes of antibodies to various epitopes which
have diverse affinities. When a disease agent attacks a vertebrate host, a whole army of different
antibodies are effective in neutralizing the disease agent. These same polyclonal antibodies can
be used in immunoassays; however, the wide-variety of antibodies make the assay less specific.
With the development of monoclonal antibodies greater specificity can be achieved in most
immunoassays.
Types of antigen-antibody reactions
Basically there are two types of serological reactions. Agglutination reactions occur between an
antibody and particulate antigens such as RBC or bacteria. Agglutination of bacteria has been a
key
method
in
identifying
and
classifying
microorganisms.
Precipitation reactions occur between an antibody and a fluid antigen. Precipitation reactions are
most useful since most antigens are soluble or can be solubilized by simple procedures. In
addition, there are many types of precipitation test which can be adapted to many types of
antigens. In many cases nonprecipitating antigen-antibody reactions can be detected indirectly
with
other
antibodies,
isotopes,
or
enzymes
or
fluorescent
dyes.
The antigen-antibody reaction takes place in two phases . In the first phase, combination of the
reactants occurs; this is followed by second phase, an aggregation (precipitation or
agglutination). The first phase stages take place almost instantaneously. In the second stage of
the reaction, aggregation of the antigen-antibody complex occurs. This phase does not occur
when monovalent antibodies or haptens are involved in the reaction. The lattice hypothesis,
which is based on multivalent antigens and bivalent antibodies, is useful in explaining the zone
phenomena. At the equivalence point all of the antigen and all the antibody molecules are
consumed in the lattice formation. With multivalent antigen where the antigenic site is not
repeated, it requires two different antibodies directed at different antigenic sites to form a lattice.
When there is an excess of antibody (prozone) , preciptitation is not observed. Each antigen is
surround by antibodies. Essentially the reverse situation occurs in ( postzone) , where too little
antibody is present to produce an aggregation each antibody molecule only binds to one antigen.
The size of the aggregation complex increases as the optimal ratio of antigen to antibody is
achieved. This is referred to as the (equivalence zone) . Antigen-Antibody binding The antigen-
antibody binding take place by noncovalent bonding between the epitope (active site on antign)
and paratope (antigen binding site on antibody). The antigen-antibody binding is strong because
of the multiple hydrogen bonds, ionic bonds, van der Waal force, and hydrophobic interactions.
The reactants must be close and their structures (antigen and antibody) must be complementary.
The reaction is reversible and neither the antigen or antibody has changed.
Antibody affinity refers to the strength of binding between the paratope of the antibody and the
epitope of monovalent antigen or hapten. The term Avidity is used to designate the strength of
binding of a multivalent antigen with an antibody possessing at least two combinding sites or
antiserum containging antibodies of differing specificities. IgM with ten binding sites or an IgG
molecule, with two binding sites, might become bound to an antigen possesing seral identical
determinants. The avidity of these Ab-Ag binding reaction is greater than the sum of the intrinsic
affinities, since all bonds must be broken simultaneously. Measurement of Antibody Affinity
The intrinsic affinity of an antibody can be expressed in terms of an affinity constant (K). This
constant can be used to express antigens affinity for an antibody. High affinity antibodies are
desirable in diagnostic, therapuetic, analytical applications. Low affinity antibodies are useful in
affinity chromatography, since the antibodies can be release with low pH. To measure the
affinity of an antibody, we must have a procedure of detecting bound and unbound ligand
(hapten). In accordance with the law of mass action, the rate of a reaction is proportional to the
concentration
of
the
reactants.
Thus,
r/c
=
Kr
+
Kn
Equation
1
Equation
1
is
a
equation
of
a
straight
line
r/c
=
Kr
+
Kn
y
=mx
+
b
In equation 1 r and c are variables, while K and n are constants.
Thus , if r/c is plotted against r, the slope will be -K, the y intercept on the vertical axis will
be Kn and the x intercept on the horizontal axis will be n, the valence.
Plotting of ligand between compartments I and II until equilibrium is reached. The difference
between the two curves reflects the percntage of total ligand bound to antibody compartment I.
Precipitation
reactions
There are many immunological assays based upon optimal precipitation of an antigen and
antibody. A few of the most commonly used methods to characterize monoclonals and
soluble antigens are: two-dimensional immunoelectrophoresis , rocket electrophoresis,,
countercurrent electrophoresis,, radial immuno-diffusion , immunoelectrophoresis , and
Western
blot,.
The radioimmuno assay is an example of how isotopes can be used to increase the sensitivity of
an immunoassay. The fluorescent antibody assay is an example of how a fluorochrome came be
used
to
increase
the
sensitivity
of
an
immunoassay.
Biotin-Avidin
Assay
Avidin is a glycoprotein derived from egg albumin, which has a very high affinity for the
vitamin, biotin, and does not bind to other substrates. Biotin can be easily coupled to IgG
antibody. Avidin can be labeled with fluorochromes, enzymes, or radioactive chemicals. If the
secondary antibody are labeled, using the biotin-avidin system, they can be efficiently employed
in
indirect
IF,
ELISA
and
RIA.
5.
Fluorescence-Activated
Cell
Sorting
Cells can now be analyzed and isolated on the basis of their distinct surface antigens, their size,
or both by a process known as flow cytometry. Flow cytometery are instruments that can analyze
properties of single cells as they pass throug an orifice at high velocity. These intruments
measure
light
scatter,
volume,
and
fluorescence.
FACS can analyze and sort lymphocyte sub populations, as identified by fluorescein-labeled
monoclonal antibody. A suspension of leukocytes is incubated with labeled monoclonal antibody
against the CD4 helper cells. The washed sample is then introduced into the sample chamber of
the cytometer and the cells are forced through a nozzle in a liquid jet. With vibration at the
nozzle tip, the stream breaks up into droplets. The size of the droplet can be regulated so that
each contains a single cell. The cells pass in front of a laser beam, which excites the fluorescent
dye (which is monitored by fluorescent detector). The droplets that emit appropriate fluorescent
signals are electrically charged in a high-voltage field between the detector and deflection plate
and separated into collection tubes. CD4 helper cells can be separated from other leukocytes
of the suspension on the basis of the fluorescence of the bound antibody.
6,Monoclonal Antibody Production.
A monoclonal antibody is produced by a single clone of B cells that has a single
specificity and is of one class or subclass. The production of monoclonal antibody begins
with the injection of a mouse with a particular antigen phases
A major advantage of this procedure is that the antigen used need not be purified, as is
the case in polyclonal procedures for producing a specific antiserum. In a few weeks,
when the B lymphocytes begin proliferating in response to the antigen, the spleen, a
primary source of these lymphocytes, is removed from the mouse. Among the many
lymphocytes isolated from this mouse spleen are a few B cells which produce the desired
antibody. These spleen cells are fused in vitro with myeloma cells, a special tumor
lymphocyte that multiplies unchecked. Like most tumor cells, these myeloma cells can
proliferate indefinitely when grown in culture. Mouse B lymphocytes and myeloma cells
are fused by adding polyethylene glycol, which acts as a chemical glue. The result is a
hybridoma, a cell that combines the antibody producing capability of a B lymphocyte
with the myeloma cell's capacity to divide and reproduce virtually forever. A
combination that assures a nearly endless source of antibodies. Among the numerous
hybridomas formed will be a few cells producing the antibody of the appropriate
specificity.
The desired cells are found and separated in a three-step process: selection, screening,
and cloning . The first step is used to select only those cells that have fused to form a
hybridoma; all unfused myeloma and spleen cells are eliminated. Hybridoma cells are
screened by one of various enzyme linked immunosorbent assay (ELISA) to identify
those hybridomas that are producing antibody molecules of the desired specificity. At this
point, the hybridoma cells are usually growing as small colonies, in separate wells of 96
well culture plates. The final step is to ensure that all cells in the culture are producing the
same antibody molecules with the desired specificity. This is done by diluting the cells
from each well so that only one cell from each well can be isolated and deposited in a
tissue culture vessel. In culture, this single cell divides, producing clones of identical
hybridoma cells. This hybridoma cell line can be used indefinitely to produce a particular
antibody in mice (ascities fluid) or tissue culture medium. These cells can be stored in
liquid nitrogen for future use. The net result is the production of monoclonal antibodies
that are uniform. A single clone of hybridoma cell will produce antibodies that are of the
same class or subclass. The monoclonal antibody will be directed against a single epitope
with the same affinity. The total time required to produce a monoclonal antibody is about
six
months.
The list of potential in vitro applications using monoclonal antibodies is vast. The only
application presented in this paper will be the use of monoclonal antibodies in
immunoassays. The major advantages of monoclonal antibodies are: (1) highly specific
antibodies can be produced in large quantities; (2) pure antigens are not necessary for
production of monoclonals; (3) clones can be frozen and reused at later date as a
standardized reagent.
The disadvantages of monoclonal antibodies are: (1) the level of technology required; (2)
the cost; (3) the time required for production; (4) and in some cases the antibody may be
too
specific
UNIT – V
Advanced techniques – automated methods – ELISA, RIA.Aplications of Nucleic acid
hybridization, PCR and blotting in diagnosis.
PART A:
1. Which of the following diseases are not transmitted by ticks:
A. Ulceroglandular tularemia
B. Bubonic plague
C. Relapsing fever
D. Lyme disease
Correct Answer: B
2. Characteristics of a bacterial capsule include:
A. All bacteria have one
B. It is composed of peptidoglycan
C. It is an important mechanism for protecting a bacteria against ingestion by PMNs
D. It is what causes the gram stain reaction
Correct Answer: C
3. As a budding basic scientist you work in a research laboratory and discover a chemical that
removes the cell wall from bacteria but leaves the organism undamaged otherwise. Things that
the bacteria will lose because of this include:
A. The ability to stain gram positive: all will look gram negative
B. The ability to move
C. The ability to have a gram stain result
D. The ability to attach to other cells
Correct Answer: C
4. Under the electron microscope you keep at home next to your MP3 player you look at a single
bacterium living in your mouth. You see a single polar flagellum stretching off from one end
looking very much like a tail. A characteristic of this organism should be:
A. An ability to stay in one place for a very long time
B. An ability to move in the direction opposite the flagellum
C. An ability to avoid ingestion by PMNs
D. An ability to induce a large antibody response
Correct Answer: B
5. On the same day the clinical laboratory identifies two strains of the same bacterial species.
One of these strains has pili on the surface; the other does not. In terms of the clinical status of
the two patients it is likely that:
A. The patient with the piliated strain is ill, the other patient is not
B. The patient with the piliated strain is not ill, the other patient is
C. Both patients are ill since pili do not correlate with virulence
D. The patient with the piliated strain is a child, the other patient is an adult
Correct Answer: A
PART B:
1.Write a note on Advanced techniques
Enzyme-Linked Immunosorbent Assay (ELISA)
One of most widely used applications of monoclonal antibodies is in enzyme-linked
immunosorbent assay (ELISA). The ELISA is a very accurate and sensitive method of detecting
antigens or haptens. The ELISA is based on antibody recognition of a particular antigenic
epitope. Monoclonal antibody-based in vitro diagnostic tests have been available commercially
since 1981, and there are now over 100 test kits available for microorganisms, hormones,
aflatoxins, drugs, tumor markers and pesticides. Monoclonal antibody technology has
encompassed an increasing array of analytes and has facilitated the development of a number of
sensitive,
inexpensive,
safe,
and
easy-to-use
assays.
Monoclonal antibodies can be cross linked to one of nine different enzymes and used in the
ELISA. Glutaraldehyde is a bifunctional cross-linker used to join the enzyme to the antigen or
antibody. Maleimide derivatives can link two separate protein molecules together, one through
an
amide
bond
and
the
other
through
a
thioether
bond.
The clinical laboratory appears to be one of the first entities to utilize monoclonal antibodies.
The ELISA is used for screening everything from drugs to the AIDS virus in humans. There are
a variety of immunoassays which make use of monoclonal antibodies and many could be used in
their present form for FDA regulatory work. An example is the two-site immunoassay developed
for Listeria spp. It employs two different monoclonal antibodies that recognize two distinct
epitopes on the same antigen. The antibodies do not compete with one another. Pure antigen is
not required for their formation, and the immunoassay is sensitive and specific. In the doubleantibody two-site immunoassay a specific monoclonal antibody directed against the antigen of
interest is passively adsorbed onto a solid phase support (polystyrene). The solid phase is then
washed to remove unadsorbed antibody. The antigen in question becomes attached to the first
monoclonal antibody. The enzyme-labeled antibody is added, which binds to a different epitope
on the already bound antigen. Following incubation, the excess labeled monoclonal antibody is
removed by washing. Substrate is added and the absorbance is measured spectrophotometrically.
The ELISA may be more sensitive than RIA. In the latter, when the radionuclide emits gamma
rays or beta particles, it is less active. In ELISA, the enzyme catalyzes a substrate molecule, but
the enzyme can be reused.
Purifying Antibodies
Purified antibodies are required for a number of techniques. Several techniques that require
purified antibodies at least in certain steps of the procedure. Many of the purified antibodies are
labeled with a tag and these labeled antibodies are then used to determine the presence of antigen
or another antibody. When labeled anti-immunoglobulins antibodies are needed it is seldom
worthwhile to prepare and label the reagents yourself. These reagents are prepared commercially
and tested by a number of companies. When the antibody is used to detect the antigen direclty;
the antibody must be purified and labeled. Directly labeling also allows two different antibodies
to be compared in the same assay by marking them with different tags. In many procedures
purified antibodies may lower the background activity of the assay. Purification may be the
easiest
way
to
concentrate
the
antibody.
There are a wide variety of methods used to purify antibodies. The correct choice of purification
will depend on a number of factors: (1) the manner in which the antibody will be used; (2) the
species in which antibody was produced; (3) the class and subclass that will be used; (4) the type
of antibody that is needed monoclonal or polyclonal antibody; and (5) the source (ascities or
tissue culture fluids) that will serve as the starting material for the purification. Table 25
summarizes the possible sources of antibodies for purification. Also included in this table are the
possible sources of antibody contamination and the expected level of purity.
Techniques that require purified antibodies.
Technique
Antibodies use
Antibody type Best source
Comments
Cell Strain
Direct Local
Anti-antigen Polyclonal-Monoclonal Prepare yourself
¡¡
Indirect local
Anti-antibody Polyclonal
Commercial
Immunoassay Direct detection Anti-antigen Monoclonal-Polyclonal Prepare yourself
¡¡
Indirect detection Anti-antibody Polyclonal
Commercial
Immunoblot Direct detection Anti-antigen Polyclonal-Monoclonal Prepare yourself
¡¡
Indirect detection Anti-antibody Polyclonal-Monoclonal Commercial
Affinity
Purification
Anti-antigen Monoclonal
Prepare yourself
Figure 25: Sources for purifying antibodies.
Source
Conc.
Type
Ab Conc.Total
Ab
Specific
Ab
Possible
Contaminating Ab
Purity
of
Specific Ab
¡¡
¡¡
(mg/ml)
(mg/ml)
¡¡
¡¡
Serum
Polyclonal
10
1%
10%
Other Serum Ab
10%
Tissue
Culture
Monoclonal
PBS 10%
1
0.05%
Calf Ab
95%
Tissue Culture
Monoclonal
0.05
0.05%
None
95%
Ascities
Monoclonal
1-10
90%
Mouse Ab
90%
to
2.Quantity and quality of purified antibodies
During the purification of antibodies, several variables need to be monitored. These include the
purity, the amount of protein, the antigen binding activity of the antibody, and specific activity
(Figure
26).
Purity At any stage, the simplest method to determine the purity of an antibody solution is to run
a portion of the sample on an SDS-polyacrylamide gel. The gel can be stained with Coomassie
blue (sensitivity 0.1-0.5 ug/band) or silver stain (1-10 ng). The following are other procedures
that can be used to check the purity of an antibody: polyacrylamide disc electrophoresis, all types
of immunoelectrophoresis, isoelectric focusing, capillary electrophoresis, ultracentrifugation, and
various types of high performance liquid chromatography HPLC. Purity is generally determined
by
more
than
one
technique.
Quantitation If the antibody is pure, you can measure the total protein concentration. A
convenient method is UV absorbance. The amount of antibody can be determined by an
absorbance measurement at 280 nm (1 absorbance unit equal 0.75 mg/ml of purified antibody). If
the antibody is not yet pure samples can be quantified by various immunological procedures.
Figures 12 -16 are procedures that can be used to quantitatively determine the concentration of
antibody in the presence of other proteins. By modifying these same procedures the class or
subclass
of
antibodies
can
be
measured.
Antigen binding activity Antibody activity is measured by comparing the purified antibody
activity to the starting material in a series of titrations. There are a wide-variety of antibody
titration procedures. A few of the most common are: precipitation, agglutination, complement
fixation, ELISA, and RIA. Some activity of the antibody preparation is generally lost during
purification but the specific activity will increase (Figure 26.).
Protein purification procedures
Purification and characterization is a prerequisite to studying all biological molecules and
antibodies are no exception. The first step in characterizing antibodies is isolation of the
molecule in pure form. There are many procedures but only those most commonly used for
antibodies will be briefly discussed. Each of these procedures will be covered in greater detail in
other lectures in this Biotechnology Seminar. Most of these procedures can be automated and
improved by HPLC technology. Most HPLC versions of the procedures will bring about a
reduction in purification time, increased resolution and better data acquisition.
Ammonium sulfate Ammonium sulfate ( (NH4)2SO4 ) precipitation is one of the most common
and oldest methods used for purifying antibodies. Proteins with exposed polar and ionic groups
form hydrogen bonds with water molecules in aqueous solutions. Proteins dissolved in aqueous
solutions have a greater affinity for water than other proteins. When a high concentration of
highly charged ions such as ammonium ions (NH4+) and sulfate ions (SO42-) are added to an
aqueous antibody solution, the ions compete with proteins for water molecules. Water is
removed and solubility of proteins is decreased which results in precipitation. Precipitated
proteins have a greater affinity for other protein molecules than they have for water molecules.
The precipitation of protein can be reversed conveniently by lowering the concentration of
ammonium sulfate by adding water. Factors that affect the concentration precipitation include
the number and position of the polar groups, the molecular weight of protein, the pH of the
solution, and temperature at which the precipitation is performed. The concentration at which
antibodies will precipitate varies from species to a species. Most antibodies will precipitate at
50%
saturation.
A disadvantage of ammonium sulfate precipitation of antibodies is that the resulting antibodies
will not be pure. They will be contaminated with other high-molecular-weight proteins, as well
as proteins that are trapped in the large flocculant precipitate. Therefore, ammonium sulfate
precipitation is not suitable for a single-step purification but must be combined with other
methods
if
a
pure
antibody
preparation
is
needed.
Caprylic acid In mildly acidic conditions, the addition of short-chain fatty acids such as caprylic
acid will precipitate most serum proteins with the exception of the IgG molecules. In
combination with other purification steps such as binding to a DEAE-matrix or ammonium
sulfate precipitation, caprylic acid will yield a relatively pure antibody preparation.
Ion exchange chromatography (IEC) Ion exchange chromatography is an application that can be
used to separate almost any type of charged molecule from large proteins to small nucleotides
and amino acids. There are two basic types of ion exchange chromatography. The first is an
anion exchanger (the matrix has positive charges) and will bind negatively charged proteins
which are at a pH above their IpH. An example of an anion exchanger is Diethyl aminoethyl
(DEAE) group. The second is a cation exchanger ( the matrix has negative charges) and will bind
positively charged proteins which are at a pH below their IpH. An example of a cation exchanger
is
carboxymethyl
(CM)
group.
Because antibodies have a more basic isoelectric point than the majority of other serum proteins,
ion exchange chromatography is a useful method for purifying antibodies. Two strategies are
commonly used. In cation chromatography, the pH is kept below the isoelectric point of
antibodies (pH 8.6). The antibody has positive charge and will bind to cation exchanger such as
CM-matrix. In the second approach the pH is raised above the IpH and the antibody has an
negative charge. The negatively charged antibody will bind to an anion exchanger such as
DEAE-matrix. As the salt concentration is raised, the antibodies will be the first of the serum
molecules to elute. Both anion and cation exchange chromatography have been used
successfully. Ion exchange is greatly enhanced when adapted to HPLC. With HPLC ion
exchange chromatography the resolution is greatly increased and the length of the run has
diminished
from
48
hr
to
30
min.
Gel filtration chromatography Gel filtration chromatography is used to separate molecules
according to molecular size. The resolution is not as high as other chromatographic techniques;
however it is found in practically all protein purification schemes. Because IgM (1,000,000)
molecules are considerably larger than IgG (150,000) and many other molecules found in serum,
gel filtration chromatography can be used to separate these two classes of molecules. To obtain a
pure preparation of IgM, gel filtration must be combined with other techniques such as
ammonium sulfate precipitation. HPLC gel filtration column are commercially available.
Immunoaffinity purification of antibodies. The only method commonly used to purify antigenspecific antibodies from a preparation of polyclonal antibodies is immunoaffinity purification. In
this procedure pure antigen is bound covalently to a solid support. The antibodies within the
polyclonal pool that are specific for the antigen are allowed to bind. The unbound antibodies are
removed by washing, and the specific antibodies are eluted with low pH (pH 2.3). This method is
unnecessary for monoclonal antibodies, which are homogeneous in their antigen binding activity.
Purification on protein A beads Chromatography of antibody solutions over a protein A bead
column is one of the most effective and widely used methods for purifying antibodies from many
types of crude preparations. Two variations on this method are given. In the first, the antibodies
are added to the column in low salt (near physiological levels). This method is applicable when
the affinity of the antibody for protein A is sufficient to allow high-capacity binding. In the
second method, the affinity of protein A for the antibody is increased by hydrophobic bonds that
form the basis for the interactions. The increased hydrophobic bonding is achieved by raising the
salt concentration in the buffer. In both variations the antibodies are eluted by lowering the pH of
the
buffer.
Protein A is a bacterial cell wall protein isolated from Staphylococcus aureus which binds to
antibodies from different animals. Protein A binds to the FC fragement of the antibody molecule.
The binding site is found in the second and third constant regions of the heavy-chain
polypeptides. Similar types of proteins are found in other bacteria, in particular streptococci from
the A, C, and G strains. The best studied of these is a polypeptide known as protein G.
3.Classing and Subclassing of Monoclonal Antibodies
Many techniques require monoclonal antibodies with specific properties. The specificity and
affinity of the antigen for antibody is a characteristic of the hypervariable region of the antibody
molecule. Because of the specificity of an antibody, closely related antigens can be distinguished
from
each
other
with
appropriate
immunoassays.
A second important property of monoclonal antibodies is found in the structure of the constant
region of the antibody. The sequence of amino acids found in the constant region of the antibody
determines the class and subclass. The different classes or subclasses will determine the affinity
for important secondary reagents such as protein A. The type of heavy and light chain can be
distinguished by simple immunochemical assays that measure the presence of the individual light
and heavy chain polypeptides. This is normally achieved by raising antibodies specific for the
different mouse heavy and light chain polypetides. The production of these antibodies is possible
because the light and heavy chains polypepetides from different species are sufficiently different
to allow them to be recognized as foreign antigens. Most often these anti-mouse immunoglobulin
antibodies are raised in rabbits as polyclonal sera, and then the antibodies specific for a particular
heavy or light chain are purified on immunoaffinity columns. Although these chain-specific
rabbit anti-mouse immunoglobulin antibodies can be made in the laboratory, it is normally easier
to
purchase
them
from
commercial
sources.
Ouchterlony classing and subclassing Originally, the Ouchterlony double-diffusion assay was the
most common method for determining class and subclass of a monoclonal antibody. They have
been largely replaced by other techniques. They are still useful when only a few tests will be run.
Samples of tissue culture supernatants are pipetted into a hole in an agar bed. Class and subclass
specific antisera are placed in other wells at equal distance from the test antibody. The two
groups of antibodies diffuse into the agar. As they meet, immune complexes form, yielding
increasingly larger complexes as more antbodies combine. When large multimeric complexes
form, the immune complexes will precipitate, forming a line of protiens that is either visible to
the
naked
eye
or
that
can
be
stained
to
increase
the
sensitivity.
Antigen-coated plates Any of the assays used to screen hybridoma cells that detect antibodies
with a secondary anti-mouse immunoglobulin can be adapted to screen for class or subclass. If
the detection method used 125I-labeled rabbit anti-mouse immunoglobulin to locate antibodies
bound to the antigen, then substituting anti-class or subclass specific antibodies for the 125I
reagent
will
identify
the
type
of
heavy
chains.
Anti-Ig antibodies One of the easiest methods for determining the class and subclass of a
monoclonal antibody is to bind class or subclass specific antibodies to the wells of a
polyvinylchloride (PVC) plate. The test monoclonal antibody is added to each well, but will bind
only to wells coated with anti-antibodies that are specific for its subclass or class. The bound
antibodies are detected using a secondary antibody specific for all mouse antibodies.
Selecting class-switch variants During the normal development of a humoral response, the
predominant class of antibodies that are produced changes, beginning primarily with IgM and
developing into IgG. These changes and others like them occur by genetic rearrangements that
move the coding region for the antigen binding site from just upstream of the IgM specific region
to the IgG region. These rearrangements help the host animal tailor the immune response to the
various types of infection. The different classes and subclasses of antibodies also have properties
that make them more or less useful in various immunochemical techniques. These differences
make the preparation of antibodies of certain classes or subclasses very valuable.
Recently, it has been shown that a process that appears similar to the natural class and subclass
switching occurs in vitro, although at a very low frequency. Therefore, any population of
hybridomas will have a small proportion of cells secreting antibodies with a different class or
subclass of antibody. The antigen binding site will be identical in these antibodies. If these cells
can be identified and cloned, then antibodies with the same antigen binding site but with
different class or subclass properties can be isolated. These shift variants generally are useful in
one of two cases, either switching from IgM to IgG or from IgG1 to IgG2a. Often these switches
are used to produce antibodies that bind with higher affinity to protein A.
When trying to identify any class or subclass switching variants, it is important to remember that
the rearrangements that occur will remove and destroy the intervening sequences, so only those
heavy chain constant regions that are found further downstream can be selected. The order of
heavy-chain constant regions is M, D, G3, G1, G2b, E, and A. Workers should also be certain
they need these variants, as the assays are tedious. It may often be more advantageous to set up
another
fusion
rather
than
isolate
switch
variants.
The most useful approach for most laboratories has been developed by Scharf and his colleagues
1985. First, a suitable assay must be developed. Because of the large number of assays that must
be performed, enzyme-linked assays are generally more useful. The assay for antibody captured
can be easily adopted by changing the detection reagent to an IgG or IgG2a specific rabbit antimouse immunoglobulin antibody.
PART C:
1. Ouchterlony double immuno diffusion
Picture of an Ouchterlony Double Diffusion plate. In this, titre value of an antigen is quantified.
Central well has an antibody and the surrounding wells have decreasing concentration of the
corresponding antigen
Ouchtherlony patterns
Ouchterlony double immuno diffusion (or agar gel immunodiffusion) is a simple, rather
dated method which is still considered to be the gold standard for detection of extractable nuclear
antigens (ENAs).
Procedure
A gel plate is cut to form a series of holes in the gel. An extract of human cells, harvested from
tonsil tissue, is placed in the center well. This extract contains a cocktail of natural human
antigens that we wish to look for. The patient's serum is then placed in one (or more) of the outer
wells and the plate left for 48 hours to develop. During this time the antigens in the tonsil extract
migrate in a radial fashion out of the center well and the antibodies in the patients serum migrate
in a radial fashion out from the peripheral wells. When the two antigen fronts meet, if there are
antibodies in the patients serum to any of the antigens in the tonsil extract, they will bind to the
tonsil antigens and form what is known as an immune complex. This immune complex
precipitates in the gel to give a thin white line.
When more than one peripheral wells are used there are many possible outcomes based on the
reactivity of the antigen and antibody selected. The zone of equivalence lines may give a full
identity (i.e. a continuous line), partial identity (i.e. a continuous line with a spur at one end), or a
non-identity (i.e. the two lines cross completely).
Theory
Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several
antigenic determinants per molecule to which antibodies can bind). Antibodies have at least two
antigen binding sites (and in the case of IgM there is a multimeric complex with up to 10 antigen
binding sites), thus large aggregates or gel-like lattices of antigen and antibody are formed.
Experimentally, an increasing amount of antigen is added to a constant amount of antibody in
solution, initially at low antigen concentration, all of the antigen is contained in the precipitate.
This is called the antibody-excess zone (i.e. prozone phenomenon). As more antigen is added,
the amount protein precipitated increases until the antigen/antibody molecules are at an optimal
ratio. This is known as the zone of equivalence or equivalence point. When the amount of
antigen in solution exceeds the amount of antibody, the amount of precipitation will decrease.
This is known as the antigen excess zone.
2.Explain about Hemagglutination.
Hemagglutination
Hemagglutination (also haemagglutination) is a specific form of agglutination that involves red
blood cells. It has two common uses in the laboratory: blood typing and the quantification of
virus dilutions.
Blood Typing
Using antibodies that bind to the A or B blood group in a sample of blood, one can determine the
blood type of the individual being tested.
For example, if antibodies that bound to the A blood group were added and agglutination
occurred, it could be concluded that the blood was either type A or type AB. Then if antibodies
that bound the B group were added and agglutination did not occur, it could be concluded that
the blood was type A.
In blood grouping the patient's serum is tested against RBCs of known blood groups and also the
patient's RBCs are tested against known serum types. In this way the patient's blood group is
confirmed from both RBCs and serum. A direct Coombs test is also done on the patient's blood
sample in case there are any confounding antibodies.
Viral Hemagglutination Assay
Many viruses attach to molecules present on the surface of red blood cells. A consequence of this
is that - at certain concentrations - a viral suspension may bind together (agglutinate) the red
blood cells thus preventing them from settling out of suspension. Usefully, agglutination is rarely
linked to infectivity, attenuated viruses can therefore be used in assays.
By serially diluting a virus suspension into an assay tray (a series of wells of uniform volume)
and adding a standard amount of blood cells an estimation of the number of virus particles can be
made. While less accurate than a plaque assay, it is cheaper and quicker (taking just 30 minutes).
This assay may be modified to include the addition of an antiserum. By using a standard amount
of virus, a standard amount of blood cells and serially diluting the antiserum, one can identify the
minimum inhibitory concentration of the antiserum (the greatest dilution which inhibits
hemagglutination).
3.Write a note on PCR.
PCR in Diagnosis of Infection: Detection of Bacteria in Cerebrospinal Fluids
The PCR is the most sensitive of the existing rapid methods to detect microbial pathogens in clinical
specimens. In particular, when specific pathogens that are difficult to culture in vitro or require a long
cultivation period are expected to be present in specimens, the diagnostic value of PCR is known to be
significant. However, the application of PCR to clinical specimens has many potential pitfalls due to
the susceptibility of PCR to inhibitors, contamination and experimental conditions. For instance, it is
known that the sensitivity and specificity of a PCR assay is dependent on target genes, primer
sequences, PCR techniques, DNA extraction procedures, and PCR product detection methods. Even
though there are many publications concerning basic protocols of a PCR assay, including DNA
extraction and preparation as well as the amplification and detection of amplicons, PCR detection of
bacteria in clinical specimens such as cerebrospinal fluid (CSF) has not yet been reviewed. Since a
variety of clinical specimens, such as blood, urine, sputum, CSF and others, vary in regard to the
nature of the content and amount available, careful design of the PCR assay for each specific specimen
before a PCR application is conducted is essential. In particular, a diagnosis based on detection of a
few bacteria in clinical specimens by using PCR must be carefully evaluated technically as well as
microbiologically. In this regard, current studies concerning detection of Chlamydia pneumoniae in
CSF obtained from patients with multiple sclerosis (MS) by using PCR provide a good example for
discussion of use of the PCR assay in diagnosis. Because C. pneumoniae is difficult to culture in vitro,
often low numbers of bacteria may be detected in the CSF of patients with chronic neurological
diseases by PCR. Therefore, in this review general PCR protocols for detection of bacteria in clinical
specimens, as well as a specific example of using PCR for detection of C. pneumoniae in CSF, will be
discussed.
4.METHODOLOGICAL ASPECTS
The PCR assay in diagnosis involves several critical steps, such as DNA extraction from specimens,
PCR amplification, and detection of amplicons. In particular, when specific clinical specimens, such
as CSF, with only a few bacteria present are tested by PCR, each procedure must be carefully designed
and performed.
CSF. CSF is widely utilized for diagnosis of diseases of the central nervous system (CNS). Because
CSF has important functions, including cushioning the brain, maintaining a constant intracranial
pressure, providing nutrients, and removing toxic metabolites from the CNS, an indirect assessment of
brain status can be obtained from the CSF. Since CSF is considered germfree, detection of microbes in
CSF, even in low numbers, provides valuable information about possible infection. However, it must
be noted that detection of microbes in the CSF does not always indicate a CNS infection, since
impairment of the blood-brain barrier may permit transit of microbes. Nevertheless, detection,
identification, and quantitation of microorganisms in CSF is important in diagnosis of meningitis and
other CNS infections. In particular, recent studies indicate possible involvement of microorganisms in
specific diseases of the CNS, including Alzheimer's disease and MS (3, 30, 45, 56). Therefore,
detection of even a few microorganisms in CSF by a standardized protocol is a critical matter for
diagnosis of such diseases. The normal adult produces approximately 500 ml of CSF per day, with
approximately 150 ml of CSF in the CNS at any given time (34). Thus, the available amount of CSF
and numbers of samplings for diagnosis are limited. Therefore, performing PCR using a CSF
specimen will become the first-line diagnostic test for CNS infections (11, 33), due to a sensitivity
requiring only a limited amount of CSF, the specificity of the assay, and speed. In fact, a number of
trials using PCR for detection of a broad range of bacteria in CSF specimens have been reported (37,
38, 42, 66). However, the sensitivity and specificity of PCR assay for detection of pathogens may not
be better than those of culture assay, which has been standardized and validated for most pathogens,
due to the dependability of PCR sensitivity on the assay process. Therefore, a negative PCR result can
be used with moderate confidence to rule out a diagnosis of infection (33).
The stability of target bacterial DNA during CSF storage is an important practical matter in clinical
laboratories. However, only limited information on the effects of various handling and storage
conditions on the stability of bacterial DNA in CSF is available. Exposure of CSF to various
environmental conditions, such as room temperature versus 4°C for up to 96 h and freeze-thawing up
to three times, does not affect the ability of a highly sensitive PCR assay to detect bacterial DNA in
CSF samples (60). That report, however, tested only limited environmental conditions. Therefore,
proper storage and handling of CSF are still essential for detection of microbes after PCR
amplification.
Contamination. Since PCR is based on DNA amplification, false-positive or -negative outcomes may
easily occur. In particular, a single PCR cycle results in very large numbers of amplifiable molecules
that can potentially contaminate subsequent amplifications of the same target sequence (39). In fact, a
primary source of false-positive reactions has been identified as carryover of amplified product from
previous reactions (41). Carryover contamination of reagents, pipetting devices, laboratory surfaces, or
even the skin of workers (35) can yield false-positive results. To control such carryover contamination,
one must prevent physical transfer of DNA between amplified samples, and between positive and
negative experimental controls. For this purpose, preparation of samples for PCR assay must be in a
room or biosafety hood separate from that in which the reactions are performed. Using a pipette tip
with an aerosol barrier is essential for avoiding cross contamination as well as carryover
contamination. UV exposure is also able to destroy contaminating amplicons but is efficient only on
surfaces and with amplicons greater than 300 bp size (19). Using uracil N-glycosylase (UNG) to
cleave the dUTP incorporated in PCR products is considered a powerful protocol to prevent carryover
amplicon contamination enzymatically (41), particularly in a clinical laboratory that is performing
PCR extensively. This is performed by substituting dUTP for dTTP and adding UNG to the master
mixture. To protect the dUTP-containing product, UNG must be inactivated chemically or by heat
before the PCR product can be analyzed further. Therefore, the dUTP protocol requires only two
changes in a standard PCR protocol: the substitution of dUTP for dTTP in all reactions and the
incubation of all PCR mixtures with UNG prior to temperature cycling. In fact, this protocol has been
applied successfully to detection of Toxoplasma gondii in CSF as well as other clinical specimens
(46).
DNA extraction. Since clinical specimens have PCR inhibitors, such as hemin, which binds to Taq
polymerase and inhibits its activity (10), DNA purification is important to avoid such effects. In fact,
inhibitors are detected frequently in CSF specimens (14), and boiling of CSF is not sufficient for
removal of inhibitors which affect the detection of microbes by PCR (9). The extraction yield of target
DNA is also a critical factor in the PCR detection of bacteria in clinical specimens, particularly when
only a few bacteria are expected to be in specimens. Since bacteria have a rigid cell wall, which may
resist an ordinary digestion protocol for DNA extraction, the extraction protocol for bacterial DNA in
clinical specimens should be an additional consideration for sample preparation.
The classical DNA extraction protocol is based on purification with organic solvents like phenolchloroform, followed by precipitation with ethanol. The precipitates obtained from CSF containing
only a few bacteria may contain too little material and may not be visible. Therefore, handling of these
precipitates may require guesswork, particularly during the washing of the precipitates. Thus, it seems
likely that the resulting yield of bacterial DNA from CSF with only a few bacteria may not be
consistent. In this regard, a recent study developed a new protocol for purification of DNA by using
solid-phase carriers, which selectively absorb nucleic acids (7). This protocol is based on the nature of
nucleic acids, which can bind to silica or glass particles in the presence of chaotropic agents such as
NaI or NaClO4 (44, 62, 65). A chaotropic extraction-glass fiber filter DNA purification (GFX;
Pharmacia Biotech, Milwaukee, Wis.) is such a protocol and utilizes a glass fiber matrix for DNA
isolation. A DNA isolation kit based on the guanidinium isothiocyanate-silica bead method (7) is also
commercially available (NucliSens isolation kit; Organon Teknika, Durham, N.C.). The NucliSens
isolation kit results in sufficient DNA yield and a highly reproducible PCR for β-globin on fixed cells
(16). However, there is no report regarding application of such kits to the isolation of bacterial DNA
from clinical specimens. Fahle and Fischer (22) examined the efficacy of viral DNA isolation from
clinical specimens, including CSF, using six different commercial DNA extraction kits. It was
concluded in the report that the NucliSens isolation kit and the Puregene DNA isolation kit (Gentra
Systems, Inc., Minneapolis, Minn.) were the most sensitive among the kits tested, including the
Generation capture column kit (Gentra Systems), MasterPure DNA purification kit (Epicentre
Technologies, Madison, Wis.), IsoQuick nucleic acid extraction kit (MicroProbe Corp., Bothell,
Wash.), and QIAamp blood kit (Qiagen, Valencia, Calf.), for extracting cytomegalovirus DNA from
clinical specimens, based on DNA recovery with the broad range of specimen types evaluated. Similar
evaluations of DNA extractions with commercial kits were also performed by three other groups (13,
36, 61). From these studies, the QIAamp kit was found to be more suitable than other commercial and
noncommercial methods evaluated for the extraction of DNA for PCR. Some commercially available
extraction and purification kits based on a solid-phase purification protocol are listed in Table 1. These
kits eliminate not only guesswork but also labor-intensive phenol-chloroform extraction steps.
However, there is only limited information regarding bacterial DNA isolation from clinical specimens,
particularly CSF, with these commercial kits.
Target genes. The choice of target genes and the design of oligonucleotide primers are critical
elements in determining the sensitivity of PCR (29, 53). Even when the same gene is selected as a
target, PCR with different primer sets shows a 100- to 1,000-fold sensitivity difference between primer
sets (29). Therefore, the sequence of primers is important in the sensitivity and specificity of PCR. The
sensitivity of PCR is also dependent on the target gene selected, because copy numbers of genes or
operons per bacterium vary. In this regard, if only the sensitivity of PCR is considered, reverse
transcription-PCR is another selection method due to the multiple copy numbers of mRNAs per
bacterium. However, the practical value of reverse transcription-PCR in diagnosis is limited due to the
short life span and the vulnerability of bacterial mRNAs.
Sequence polymorphism of a target gene is another concern in regard to PCR specificity. Some
bacterial genes, such as the C. trachomatis outer membrane protein gene, have hypervariable regions
within the gene (23). Therefore, PCR products of different sizes as well as different sequences may
occur between clinical isolates of the bacterium when such a gene is selected as a target for PCR.
A universal PCR that amplifies conserved regions in various bacteria is ideal to detect any pathogen in
screening of clinical specimens (8, 40, 42, 49, 63). For this purpose, the conserved region of the 16S
rRNA gene has been selected as a target gene for the universal PCR due to the fact that almost all
common bacterial pathogens found in body fluids have been sequenced (27, 42, 49). Utilizing this
universal PCR, a high detection sensitivity of PCR for 10 gram-negative and 250 gram-positive
bacteria in CSF has been reported (42). However, since the universal PCR can detect almost all
bacteria, including normal flora such as staphylococci on the skin, discrimination for contaminants is
difficult, particularly when specimens contain few bacteria.
PCR protocols. There are several PCR protocols to enhance sensitivity, especially when dealing with
small numbers of bacteria as the target. Nested PCR is one of these protocols for detection of only a
few bacteria in clinical specimens. The process utilizes two consecutive PCRs. The first PCR contains
an external pair of primers, while the second contains either two nested primers that are internal to the
first primer pair or one of the first primers and a single nested primer. The larger fragment produced
by the first reaction is used as the template for the second PCR. The sensitivity and specificity of DNA
amplification can be considerably improved by using such nested PCR, sometimes with 1,000 times
more sensitivity than a standard PCR. However, in the case of detection of C. pneumoniae by nested
PCR in a standard solution spiked with bacteria, sensitivity was not always improved compared with
that of a standard single PCR. For example, nested and single PCRs with primers specific for the C.
pneumoniae omp-1 gene showed the same sensitivity (0.005 inclusion or 2.5 elementary bodies per
PCR) (2).
A frequently encountered problem in PCR amplification of target gene sequences is the appearance of
spurious smaller bands in the product spectrum (17). This is usually interpreted to be due to
mispriming by one or both of the oligonucleotide amplimers to the target template. Touchdown PCR is
designed to avoid such problems and provides a clearly specific PCR band. The touchdown PCR
utilizes a protocol with decreasing annealing temperatures at every cycle from above to below the
expected annealing temperature (17). The application of this technique to detection of C. pneumoniae
provided an improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit per PCR) (43).
Detection of PCR products. There are several different detection protocols reported for PCR
products besides the traditional electrophoresis method on an ethidium bromide-containing agarose
gel. Southern hybridization with a specific probe labeled with a radioisotope or nonradioisotope
marker to PCR amplicons has been widely utilized for the study of PCR specificity. This detection
protocol also provides a higher sensitivity than the ethidium bromide detection method but requires
extra blotting and hybridization steps. The digoxigenin (DIG)-PCR-enzyme-linked immunosorbent
assay (ELISA) is one of the PCR amplicon detection methods utilizing a microtiter plate and is now
commercially available (Roche Molecular Biochemicals, Indianapolis, Ind.). This method involves
capture amplicons labeled with DIG by the probe immobilized onto the surface of a streptavidincoated ELISA plate. The bound hybrid is detected with an anti-DIG-peroxidase conjugate and the
colorimetric substrate. This ELISA system has been shown to be 10 to 100 times more sensitive than
the traditional electrophoresis method (48). The PCR-immunoassay detection method utilizing a
special small device (Clearview Immunoassay Detection Device; Oxoid Inc., Ogdensburg, N.Y.),
which holds a membrane and a sample application pad containing latex beads labeled with an anti-2,4dinitrophenol antibody, is another type of detection method for amplicons designed to detect specific
bacteria in clinical isolates (12, 59). The membrane utilized in this system is coated with lines of
antibiotin antibody and anti-DIG antibody. Therefore, an evaluation of PCR results as positive or
negative by utilizing this detection kit in clinical laboratories which do not have electrophoresis
equipment is relatively easy. The application of this kit for detection of Neisseria meningitidis in CSF
showed a detection limit of one to three organisms per PCR, which is 10 times more sensitive than
detection of PCR products on traditional electrophoresis with agarose gels (54). Fluorescent probebased assays with labeled primers or specific probes labeled with a fluorescent dye have been
developed with the advantages of a closed system that avoids carryover contamination during the PCR
and increased detection sensitivity for amplicons. There are two types of assays, using real-time and
end point readings. Particularly the real-time PCR, which provides quick and accurate information
regarding target genes, has been increasingly utilized. This approach has the advantage of quantitating
the PCR in the exponential phase rather than using the end point accumulation of PCR product or
trying to capture the PCR in the exponential phase, as was done previously in many quantitative PCRs
(52). This non-gel-based technique has several other advantages over ordinary agarose gel-based
techniques. For instance, this system allows a large increase in throughput. The fluorescent-probe
assay is run in a 96-well format, and many of the steps in the assay are automated. The assay is a
closed system in which the reaction tube is never opened after amplification. In addition, it uses an
automated detection system that quantitates and calculates the degree of fluorescence over that for the
control at each cycle and hence accurately defines the cycle number and linear range for a positive
result (52). Even though presently there are few reports on detection and quantitation of bacteria in
CSF by real-time PCR, this technique has excellent potential as a major protocol for PCR detection of
bacteria in clinical specimens, including CSF, due to these advantages.
5.DETECTION OF C. PNEUMONIAE IN CSF BY PCR
C. pneumoniae is an obligate intracellular bacterium responsible for a variety of respiratory illnesses,
including 10% of community-acquired pneumonias, bronchitis, pharyngitis, and sinusitis.
Seroepidemiologically, 50 to 80% of the adult population has been shown to have prior exposure to
this pathogen (4, 26, 57). Furthermore, recent studies have revealed that this bacterium may be
involved in some chronic inflammatory diseases, such as asthma (28), arthritis (25), atherosclerosis
(47), Alzheimer's disease (3), and MS (56). Since the culture of C. pneumoniae is difficult in most
clinical laboratories, determination of this bacterium in clinical specimens has been widely performed
using the PCR technique even though there is no standardized PCR method for detection of this
organism (2). Therefore, PCR results with clinical specimens to detect this bacterium vary widely (2).
MS is a chronic demyelinating disease of the CNS characterized by focal areas of demyelination.
Although the exact etiology of MS is unknown, it is generally accepted that autoimmunity is involved
and that the autoantigen(s) probably resides in CNS myelin, the target of the immune response (1). In
this regard, current studies argue for an infectious agent as an initiating or enhancing factor for MS
with any immunological mechanisms (24). To identify a specific causative agent for MS, many groups
have attempted to detect microbes in CSF as well as lesions of CNS obtained from MS patients.
However, there have been no consistent results with specific pathogens. Recent studies by Sriram et al.
(56) highlighted a possible involvement of a bacterium in MS with the finding of C. pneumoniae in the
CSF of nearly all patients with MS but in only a small proportion of CSF samples from control
subjects without MS by utilizing PCR and culture methods. That study has shown the highest
association with MS of any organism to date. However, other research groups were not successful in
detecting this bacterium or found only a low rate of detection of C. pneumoniae in CSF from MS
patients (Table 2).
To date, there have been 10 reports concerning detection of C. pneumoniae in CSF from MS patients
by PCR (Table 2). The results of studies concerning the presence of C. pneumoniae in CSF of MS
patients as determined by PCR have shown a very wide variation in the positive rate, ranging from 0%
to almost 100%. Such variation of the C. pneumoniae positive rate in CSF may be dependent on the
source of CSF and/or the PCR protocol utilized. Since there is no standard PCR protocol for C.
pneumoniae detection and no consistent pattern of positive results among the various laboratories
determined by a multicenter comparison trial of PCR assays for detection of C. pneumonia.
C. pneumoniae DNA extraction. Five of the 10 published papers mentioned above were letters;
therefore, details of DNA extraction and the PCR protocols utilized in these studies were not fully
described. For instance, Numazaki and Chiba (K. Numazaki and S. Chibar, Letter, Neurology 57:746,
2001) and Pucci et al. (E. Pucci, C. Taus, E. Cartechini, M. Morelli, G. Giuliani, M. Clementi, and S.
Menzo, Letter, Ann. Neurol. 48:399-400, 2000) did not provide information as to the amount of CSF
tested. The amount of starting material as well as the final volume of DNA solution appears to affect
the sensitivity of overall detection. Therefore, whether the protocol utilized is sensitive enough or not
cannot be evaluated due to the lack of information in such reports. Only two reports, which were full
papers, described the amount of CSF tested, the DNA extraction protocol, the final volume of the
DNA solution, and the final concentration of CSF used for PCR. For instance, Ikejima et al. (31)
extracted DNA in 50 μl from 200 μl of CSF, which means a fourfold concentration of the original CSF
volume. In the work of Sriram et al. (56), DNA was extracted in 20 μl from 300 μl, a 15-fold
concentration of CSF. It is apparent that these different concentrations of CSF may affect the final
sensitivity of the PCR assay. None of the reports except those of Ikejima et al. (31) and Sriram et al.
(56) provided the final volume of DNA extracted.
Most studies utilized the DNA extraction protocol with solid-phase carriers, such as Qiagen columns
that hold a silica gel membrane. In only two studies, those of Sriram et al. (56) and Pucci et al. (letter),
was DNA extraction performed by a standard extraction protocol such as phenol-chloroform and
ethanol precipitation. As mentioned above, the use of solid-phase carriers for DNA isolation may
contribute to a consistent DNA extraction yield, particularly with CSF, which may not contain many
leukocytes or microbes in a sample if the patients do not have meningitis.
It is notable that only one report (31) considered the extraction protocol for bacterial DNA. C.
pneumoniae is a gram-negative bacterium and has lipopolysaccharide and other outer membrane
components in its cell wall, which contribute to osmotic stabilities as well as to rigidity, particularly of
elementary bodies, the infectious form that resists physical and chemical pressures in the extracellular
environment. Therefore, the procedure for extraction of C. pneumoniae DNA from clinical specimens
must be designed for bacterial DNA extraction, particularly for specimens that may have only a few
bacteria, such as CSF from MS patients. In fact, the study showed that when two extraction protocols
were examined, one designed for extraction of mammalian DNA from blood samples and one
designed for extraction of bacterial DNA, the protocol for bacterial DNA extracted the microbial DNA
more efficiently (31).
PCR method. The major outer membrane protein (MOMP) genes, such as omp-1 (ompA) (32), of C.
pneumoniae have been utilized widely in PCR as a target gene for detection of this bacterium. C.
pneumoniae has many outer membrane proteins, including cysteine-rich proteins OmcA and OmcB
(20, 21) as well as the MOMP encoded by omp-1. It is known that C. trachomatis MOMP has genetic
variation, including in amino acid sequences (58), but not much information regarding C. pneumoniae
MOMP is available. Therefore, the design of primers for MOMP genes must be undertaken with
special care. The species-specific region of the 16S rRNA gene is also frequently utilized as a target
gene in PCR for detection of C. pneumoniae (15, 18; S. A. Morre, C. J. De Groot, J. Killestein, C. J.
Meifer, C. H. Polman, P. Van der Valk, and A. J. Van den Brule, Letter, Ann. Neurol. 48:399, 2000).
In this regard, it is noteworthy that the detection sensitivities of the two PCRs with omp-1 versus 16S
rRNA gene primers under each set of optimal conditions were different (31). The PCR for omp-1 was
at least 10 times more sensitive than that for the 16S rRNA gene. Furthermore, when both PCRs were
used for detection of C. pneumoniae in CSF obtained from MS patients, the PCR for the 16S rRNA
gene could not detect any C. pneumoniae DNA, even though the PCR for omp-1 detected C.
pneumoniae DNA in the same CSF samples. Whether the design of primer sequences or the selected
target gene is responsible for the different sensitivities of these PCRs is unknown, but it is apparent
that both primer design and choice of target gene for PCR for detection of C. pneumoniae in CSF are
important in the detection of C. pneumoniae by PCR.
It is generally accepted that nested PCR may be more sensitive than single PCR due to the utilization
of two consecutive PCRs. However, in practice, nested PCR does not always give a higher sensitivity
than single PCR (2). In addition, nested PCR is much more prone to contamination. Therefore,
detection of C. pneumoniae in CSF which may not contain many bacteria by nested PCR must be
carefully performed; otherwise, no other method presently can confirm positive PCR results.
Detection of C. pneumoniae in CSF. Controversy surrounds the detection of C. pneumoniae in CSF
obtained from MS patients, primarily because of the lack of a definitive test for detecting the small
numbers of C. pneumoniae present. Culture is always considered the “gold standard” in microbiology
but is difficult to perform for certain fastidious bacteria such as C. pneumoniae in specific clinical
specimens. For instance, this bacterium has not been successfully cultured from blood samples,
although its DNA can be detected in blood and the organism has been recovered in limited numbers
from vascular tissue specimens (18). Even though PCR enables the detection of low concentrations of
bacteria in clinical specimens, great variability of detection is usually found in CSF from MS patients
(Table 2), atherosclerotic tissue samples, and peripheral blood mononuclear cells, ranging from a 0 to
100% detection rate between studies (5, 6, 64). In this regard, a recent study conducted by Smieja et al.
(55) demonstrated the relationship between target concentrations and PCR detection rate; that is, lower
concentrations of C. pneumoniae were only intermittently PCR positive, and this relationship was
predictable from a statistical viewpoint. From this point of view, theoretically a larger number of
replicates of a PCR assay may result in a better chance for detecting low numbers of bacteria. In other
words, the negative PCR results obtained from a single PCR test may not be a true negative due to the
low validity of detection with a lower concentration of target. Since none of the papers reporting
results of C. pneumoniae detection by PCR in CSF from MS patients provided any replicate number of
PCR tests, the negative results reported may possibly not be true negatives but could indicate that
there were few bacteria, if any, present.
CONCLUSIONS
It has been well documented that specific infectious agents may be involved in autoimmune
diseases, such as Trypanosoma cruzi as the causative agent of Chagas' disease and Streptococcus
pyogenes and measles virus for encephalomyelitis (51). Some chronic inflammatory diseases which
are not yet definitely classified as an autoimmune diseases are also considered to be linked to some
microbial infections. However, in contrast to infectious diseases, causative or contributing microbes
for such chronic inflammatory diseases, including autoimmune diseases, may not be readily detected
in specimens obtained from the specific lesion. Since chronic inflammatory diseases as well as
autoimmune diseases, including atherosclerosis and MS, may be attributed to the immune response
through molecular mimicry and/or the possible adjuvant effect of infectious agents (50), the presence
of microbes in a lesion may not always be necessary. Even under such circumstances, a consistent
detection of bacteria in specimens should be critical in diagnosis and future therapy. In this regard,
PCR is the most reliable assay for detection of microbes in clinical specimens. Careful design and
protocol for a PCR assay to detect, measure, and identify microbes in clinical specimens are essential.
Analysis of PCR results is also a critical issue in diagnosis, particularly for chronic inflammatory
diseases. Application of a clinically relevant PCR assay to these issues in monitoring bacterial
presence in CSF may reveal a role for bacteria, such as C. pneumoniae, in chronic inflammatory or
autoimmune diseases, such as MS.
6. Write a note on Protein detection technique.
Western Blot
In these assays, antigens are first electrophoresed and then blotted onto a piece of
filter-paper. Thus the filter paper is the solid phase to which the antigen is bound.
The filter is blocked with BLOTTO and then reacted with appropriate “tagged”
antibodies and then substrate. As above, a color reaction indicates a “positive”
test.
7. WIDAL TEST
INTRODUCTION:
Enteric fever specific agglutinins (antibodies) are detected in patients after 15 days of fever.
BCG vaccinated patient’s serum may show elevated titre of all three ‘H’ agglutinins. Stained
salmonella antigens are used to detect and identify specific antibodies in serum samples from
patients suffering from enteric fever.
PRINCIPLE:
Bacterial suspension which carry antigen will agglutinate on exposure to antibodies to
salmonella organisms.
SAMPLE :
0
Fresh seurm is preferred. In case of any delay performing the test, serum should be stored at 2 0
8 C.
STORAGE & STABILITY OF REAGENTS:
0
0
All reagents are ready to use and stable at 2 – 8 C till the expiry date.
REAGENTS:
1. Antigen suspension, S. typhi O.
2. Antigen suspension, S. typhi H.
3. Antigen suspension, S. paratyphi ‘AH’.
4. Antigen suspension, S. paratyphi ‘BH’.
5. Polyspecific positive cotrol
6. Glass Slides with 6 reaction circles and Mixing sticks.
Bring all reagents to Room Temperature before testing. Shake well antigens before actual use.
PROCEDURE:
SLIDE TEST
1. Place one drop of positive control on one reaction circles of the slide.
2. Pipette one drop of Isotonic saline on the next reaction cirlcle. (-ve Control)
3. Pipette one drop of the patient serum tobe tested onto the remaining four reaction circles.
4. Add one drop of Widal TEST antigen suspension ‘H’ to the first two reaction circles. (PC &
NC)
5. Add one drop each of ‘O’, ‘H’, ‘AH’ and ‘BH’ antigens to the remaining four reaction circles.
6. Mix contents of each circle uniformly over the entire circle with separate mixing sticks.
7. Rock the slide, gently back and forth and observe for agglutination macroscopically within
one minute.
INTERPRETATION OF RESULTS:
Agglutination is a positive test result and if the positive reaction is observed with 20 ul of
test sample, it indicates presence of clinically significant levels of the corresponding antibody in
the patient serum.
No agglutination is a negative test result and indicates absence of clinically significant levels of
the corresponding antibody in the patient serum.