Out lines
• Clinical assessment
• •Collecting and transporting specimens
• •Microscopy
• •Culture
• •Sensitivity
• •Non-cultural diagnostic methods
• •Virological diagnosis
• •Interactions between humans and microbes
• •Normal flora
Some basic principles of microbiology testing
A crash course in microbiology
Follow a specimen through the lab
Laboratory staffing issues
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Major group of the microbial world
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Bacteria
Archeae
Fungi
Algae
Parasite
Viruses
Major features
Small
Diverse (appearance, genetics)
Diagnosis of Bacterial infections
• COLLECTION OF SPECIMENS (Sample):
1. First, Determine agents of the disease.
• 2.Choose the appropriate specimen.
• 3.Obtain specimen properly, avoiding
contamination.
• 4.Transport quickly to lab.
• 5.Store properly
• 6.Provide all information needed by lab.
Staff.
BACTERIOLOGICAL
METHODS:
• Naked eye examination.
• Microscopical exam.
• Specimen culture .
4. Isolate identification by:
a)Biochemical reactions.
b)Growth on selective media
c)Anti-sera testing.
d)Microscopical examination.
5. Sensitivity testing of
isolate.
6. Serological tests to
detect antibodies.
7. Isolate typing for
epidemiological studies,
e.g.: phage typing.
BLOOD CULTURE:
1. Diseases suspected:
septiceamia, endocarditis,
osteomyelitis, meningitis,
pneumonia, enteric fever,
brucellosis, etc.
2. Organisms suspected:
S. aurous, S.pneumoniae, E.coli,
K.pneumoniae, P.aeruginosa,
S.typhi, Brucella, etc.
3. Collect at least:
10 ml. blood every 24 hours,
because bacteria in blood are
scanty and intermittent.
4. Collect blood:
aseptically to avoid contaminants.
5. The blood culture bottle
obtained from the lab. must
contain 100 ml. of a suitable
growth medium, e.g.: brainheart infusion broth.
6. In the lab., blood culture
bottles are checked daily
for turbidity up to 21 days.
7. Bottle is subcultured
after 24 hrs., 72 hrs, one
week, 2 weeks, and 3
weeks.
Standard media used for
subculture are:blood agar,
McConkey, chocolate agar,
Sabouraud agar, etc.
8. Identification of isolate
is by standard methods
mentioned above, and
sensitivity tests are
performed.
9. If no growth after three
weeks, discard bottle.
THROAT CULTURE:
1.Mainly used to isolate ßhaemolytic S.pyogenes
that causes pharyngitis.
Requested to diagnose
diphtheria, gonorrhea, &
candidosis.
2. Swab posterior pharynx,
tonsils, & tonsillar fossae.
3. Swab is inoculated on B.A. and
bacitracin disc is added. Then
incubate for 18-24 hrs at 37˚C.
4. Colonies of gp. (A) S.pyogenes
are ß-haemolytic and bacitracin
sensitive.
SPUTUM CULTURE:
1.Performed to diagnose
pneumonia, TB, lung abscess.
2.Sputum must be real not saliva.
3.Gram stain will show if it is
saliva or not. Good sputum shows
(25) leucocytes and less than
(10) epithelial cells per 100x
field.
5. Do gram stain to assess
cause of pneumonia (large
numbers of organisms) .
6. Culture is made on B.A. and
other selective media.
Identify by serological and
biochemical tests.
4. If the patient cannot
cough you may choose:
a) Induction of sputum.
b)Transtracheal
aspirate
c) Bronchial lavage.
d) Lung biopsy.
SPINAL FLUID CULTURE:
1.CSF is collected to diagnose
meningitis, encephalitis,
brain abscess, subdural
empyema.
2.Causes of meningitis are:
(3 encapsulated organisms)
N.meningitidis, S.pnuemoniae,
and H.influenzae.
7. Mycoplasma is
diagnosed by antibody
rise on serology. TB is
diagnosed by ZN stain
and culture on L.J
medium.
3. Send specimen immediately to
laboratory. Gram stain may give a
presumptive diagnosis.
4. Identification is made by antisera
and capsule swelling reaction, and
immunofluorescence.
5. Culture is on B.A. & chocolate agar.
Incubate plates at 35˚C in 5% Co2 .
6. Myco.TB & Cryp.neoformans
cause subacute meningitis. ZN
is made to identify Myco.TB .
Cryp.neoformans capsule may
be detected by India Ink
staining.
7. Heamatin (X-factor) and
NAD (V-factor) may help in
identification of H.influenzae.
8. Serological tests (latex
agglutination) are used to
identify organism causing
meningitis.
STOOL CULTURE:
1. Pathogenic organisms are
Shigella, Salmonella,
Campylobacter.
2. Stool general may reveal:
a) Leukocytes and pus cells
by methylene blue stain.
b) Gram stain is not
performed
3. Culture on McConkey & Eosinmethylene blue, & other
selective media. Identify by
biochemical reactions and
antisera. Widal test is made for
enteric fever.
4. C.jejuni is cultured on selective
Skirrow agar at 42˚C in 10% Co2
URINE CULTURE:
1. Performed to diagnose
pyelonephritis & cystitis.
Organisms isolated are:
E.coli, Prot., Enterobacter
E.faecalis, Pseud., Klebs.
2. Midstream, morning urine sample is
collected after washing external
orifices. Suprapubic aspiration and
catheterization may also be used
for urine collection.
3. If there is delay culture urine
within one hour after collection, or
store at 4˚C for no more than 18
hrs.
4. Bacterial urine counts
are made by inoculating
the sample on McConkey
agar using a 0.001(1µl) loop
Then multiply number of
3
colonies by 1000 (10 ).
@ Count interpretation :
a)For symptomatics:
significant count is 100
×
3
10 colonies/ml.
b) For asymptomatics:
significant count is only
100 colonies /ml.
GENITAL TRACT
CULTURE:
1. Performed to diagnose
gonorrhea caused by
N.gonorrheae, using
culture and microscopical
examination.
2. Discharge is swabbed from
urethra, cervix, & anal
canal.
It is inoculated quickly on
Thayer-Martin, chocolate
agar, or transported in
trans-grow or Stuart media.
3. N.gonorrheae is identified
microscopically as gram
negative intracellular,
diplococci within the pus
(neutrophil) cell.
4. C.trachomatis may cause NSU.
It is cultured on yolk sac of
chick embryo or human tissue
culture.
5. Syphilis T.pallidum is
seen by D-F microscopy of
a chancre fluid.
Syphilis is diagnosed by
non-specific VDRL, RPR &
specific TPHA, FTA-ABS
serological tests.
WOUND AND
ABSCESS
CULTURES:
1. Abscess is caused by
Bacteroides, S. aurous,
S.pyogenes.
Wound infections are due to
C.perfringes, S.aurous,
2. Swab is transported
immediately to lab. in
thioglycolate or RCMM.
Several aerobic and
anaerobic media are
inoculated.
SEROLOGICAL METHODS:
There are two methods:
1.Identification of an organism with
known antiserum:
Capsular swelling(Quelling)
reaction: The capsule swells up
when comes in touch with specific
antiserum. Reaction is positive with
S.pneumoniae, H.influenzae,
N.meningitidis.
Slide agglutination
test:
Used to identify
Salmonella, & Shigella,
looking for O, H, & Vi
antigens.
Latex agglutination test:
Latex beads are coated with
specific antibody, and
agglutinated by homologous
antigen. The test is used in
diagnosis of H.influenzae,
N.meningitidis, Cryptococcus
neoformans.
Counter immunoelectrophoresis
test:
The unknown bacterial antigen
and the known specific antibody
move towards each other and
form a precipitate. The test is
used to diagnose CSF pathogens,
e.g.: H.influenzae, N.meningitidis,
S.pneumoniae.
ELISA: an enzyme is linked to
the known antibody and used to
detect the homologous antigen.
Fluorescent – antibody test:
the known antibody is labeled
with a fluorescent dye &
detected by an U.V.microscope,
either directly or indirectly
when antibody unites with
antigen.
2. Identification of serum
antibodies with known antigens:
Slide & tube agglutination
test
Serial 2-fold dilution is made
for patient serum and then
bacterial antigen is added.
Highest dilution of serum with
agglutination shows the titre.
This test is to diagnose:
enteric fever, brucellosis,
plague, leptospirosis and
rickettsial diseases.
Serological tests for
syphilis: Include:
@ Non-treponemal tests:
using cardiolipin antigen:
Rapid plasma regain (RPR) and
VDRL tests.
@ Treponemal tests: include
TPHA and FTA-ABS tests.
Cold agglutinin test:
Patients infected with
Mycopl. pneumoniae will
develop autoimmune
antibodies that agglutinate
human RBC at 4˚C but not
at 37˚C.
Diagnostic methods in Microbiology
Task of the methods – to make the
microorganisms visible and measurable
Cultivation
Bio testing
Immunological methods
Biochemical methods
Microscopy
Molecular methods
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Methods
Morphology
Staining properties
Growth on different laboratory conditions and
media
Phenotyping protocols use purely biological
phenomena and usually refer to study of
proteins
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Lack of reproducibility •
Poor discriminatory power •
Difficulties in typing •
Genotyping methods
Genetic methods generally seek to detect
polymorphism at the level of nucleic acid
Genotypes are more specific, more easily,
quantified and standardized among the
different organisms
The genome is unique in individual
Several molecular methods in a chosen
genetic marker are commonly used to target
the genome or the organism
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Restriction endonuclease
T was discovered that a type of bacterial •
enzyme was found to have the ability to cut
DNA in test tube
These restriction endonuclease, so named •
because they cut double stranded DNA at
restricted site
PCR
Helps rapid detection •
Laboratory Investigation of Microbial infections
Examining specimens to detect isolate and identify pathogens:
1- Microscopy
2- Culture techniques
3- Biochemical reactions
4- Serological identification:
5- Molecular biology techniques
6- Bacteriophage typing
1- Microscopy
Microorganisms can be examined microscopically for:
a- Bacterial motility:
Hanging drop method:
A drop of bacterial suspension is placed between a cover slip and glass slid
b- Morphology and staining reactions of bacteria:
Simple stain: methylene blue stain
Gram stain: differentiation between Gm+ve and Gm–ve bacteria
. Primary stain (Crystal violet)
. Mordant (Grams Iodine mixture)
. Decolorization (ethyl alcohol)
. Secondary stain ( Saffranin)
Ziehl-Neelsen stain: staining acid fast bacilli
. Apply strong carbol fuchsin with heat
. Decolorization (H2SO4 20% and ethyl alcohol
. Counter stain (methylen blue)
2- Culture Techniques
* Culture media are used for:
- Isolation and identification of pathogenic organisms
- Antimicrobial sensitivity tests
* Types of culture media:
a- Liquid media:
- Nutrient broth: meat extract and peptone
- Peptone water for preparation sugar media
- Growth of bacteria detected by turbidity
b- Solid media:
- Colonial appearance
- Hemolytic activity
- Pigment production
Types of solid media
1- simple media:
Nutrient agar
2- Enriched media: media of high nutritive value
. Blood agar
. Chocolate agar
. Loffler’s serum
3- Selective media: allow needed bacteria to grow
. Lowenstein–Jensen medium
. MacConkeys agar
. Mannitol Salt Agar
4- Indicator media: to different. between lact. and non lact. ferment
. MacConkeys medium
. Eosine Methlyne blue Agar
5- Anaerobic media: for anaerobic cultivation
. Deep agar, Robertson’s Cooked Meat Medium
Colonial appearance on culture media
. Shape . Size
* Colony morphology:
. Edge of colony
. Color
* Growth pattern in broth:
. Uniform turbidity
. Sediment or surface pellicle
* Pigment production:
. Endopigment production (Staph. aureus)
. Exopigment production (Ps. aeruginosa)
* Haemolysis on blood agar:
. Complete haemolysis
(Strept. Pyogenes)
. Partial haemolysis
(Strept. Viridans)
* Growth on MacConkey’s medium:
. Rose pink colonies
(Lactose fermenters)
. Pale yellow colonies
(Non lactose fermenters)
3- Biochemical Reaction
Use of substrates and sugars to identify pathogens:
a- Sugar fermentation:
Organisms ferment sugar with production of acid only
Organisms ferment sugar with production of acid and gas
Organisms do not ferment sugar
b- Production of indole:
Depends on production of indole from amino acid tryptophan
Indole is detected by addition of Kovac’s reagent
Appearance of red ring on the surface
e- H2S production:
Depends on production H2S from protein or polypeptides
Detection by using a strip of filter paper containing lead acetate
3- Biochemical Reaction (cont.)
c- Methyl red reaction (MR):
Fermentation of glucose with production of huge amount of acid
Lowering pH is detected by methyl red indicator
d- Voges proskaur’s reaction (VP):
Production of acetyl methyl carbinol from glucose fermentation
Acetyl methyl carbinol is detected by addition KOH
Color of medium turns pink (positive)
e- Action on milk:
Fermentation of lactose with acid production
Red color if litmus indicator is added
3- Biochemical Reaction (cont.)
f- Oxidase test:
Some bacteria produce Oxidase enzyme
Detection by adding few drops of colorless oxidase reagent
Colonies turn deep purple in color (positive)
g- Catalase test:
Some bacteria produce catalase enzyme
Addition of H2O2 lead to production of gas bubbles (O2 production)
h- Coagulase test:
Some bacteria produce coagulase enzyme
Coagulase enzyme converts fibrinogen to fibrin (plasma clot)
Detected by slide or test tube method
i- Urease test:
Some bacteria produce urease enzyme
Urease enzyme hydrolyze urea with production of NH3
Alklinity of media and change color of indicator from yellow to pink
4- Animal pathogenicity
* Animal pathogenicity test:
Animals commonly used are guinea pigs, rabbits, mice
* Importance of pathogenicity test:
- Differentiate pathogenic and non pathogenic
- Isolation organism in pure form
- To test ability of toxin production
- Evaluation of vaccines and antibiotics
Serological identification
A- Direct serological tests:
- Identification of unknown organism
- Detection of microbial antigens by using specific
known antibodies
- Serogrouping and serotyping of isolated organism
B- Indirect serological tests:
- Detection of specific and non specific antibodies
(IgM & IgG) by using antigens or organisms
Molecular Biology Techniques
A- Genetic probes (DNA or RNA probes):
Detection of a segment of DNA sequence (gene) in unknown
organism using a labeled probe
Probe: consists of specific short sequence of labeled singlestranded DNA or RNA that form strong covalently
bonded hybrid with specific complementary strand of
nucleic acid of organism in question
B- Polymerase chain reaction (PCR):
Amplification of a short sequence of target DNA or RNA Then
It is detected by a labeled probe
C- Plasmid profile analysis:
Isolation of plasmids from bacteria and determination of their
size and number compared with standard strains by agarose
gel electrophoresis
Practical applications using phages
* Phages are important as a research tools
* Phages are used as vectors in DNA recombinant
technology
* Phage typing of bacteria is important in tracing
source of infection for epidemiologic purposes
Direct and Indirect Testing
Direct: Demonstration of the presence of an •
infectious agent
Culture –
Microscopy –
Molecular methods such as PCR –
Indirect: Demonstration of presence of antibodies to •
a particular infectious agent
Serology –
Sterile versus Non-sterile Body Sites
Sterile body sites: •
These sites normally do not contain any bacteria, so any –
bacteria found there are significant
Blood •
Spinal fluid •
Non-sterile body sites: •
These sites are open to the external environment and –
normally contain bacteria
Throat •
Feces •
Specimens from Sterile Sites
Any organism growing in a normally sterile •
site is significant
Identify it •
Specimens from Non-Sterile Sites
Only look for specific pathogens •
Physician will order test for a specific •
organism, or group of organisms
Other “normal flora” bacteria will be •
present, but are not be identified
Sensitivity
The fraction of those with the disease •
correctly identified as positive by the test.
Isolation and identification of a known •
pathogenic organism may not be a very
sensitive test
If the organism is present, it may not be found –
100% of the time
There can be false negatives •
Documentation
Specimen is logged in upon arrival in laboratory
All tests and results are recorded and initialed by
microbiologist
All media and reagents are batch tested with positive
and negative controls
All equipment is checked at least once a day to be
sure it is operating within predetermined parameters
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Specimen
Appropriateness
Collection
Transport to lab
Inoculation of media
Culture and isolation
Confirmation
Report
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Transport to Laboratory
Safe packaging •
Good labeling •
Temperature •
Inoculation of Media
Use appropriate culture media •
What kind of specimen is it? –
What test did the physician request? –
Culture media
Used to grow bacteria •
Can be used to: •
Enrich the numbers of bacteria –
Select for certain bacteria and suppress others –
Differentiate among different kinds of bacteria –
Identification of Microorganisms
Microscopic examination
Culture characteristics
Biochemical reaction
Nucleic acid analysis
Serological tests
Patients symptoms
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Classical bacterial identification can only be
performed on pure cultures of bacteria
(ideally, all descendants from one bacterial
cell)
Isolation of Individual Bacteria
Specimen is “streaked”, using a sterile loop, •
onto solid media.
The agar plates (media) are incubated at •
appropriate temperature and atmosphere
Often at 35º C. –
Often at 5% CO2 –
Usually first examined after 24 hours –
“Streaking a Plate”
Classical bacterial identification can only be
performed on pure cultures of bacteria
(ideally, all descendants from one bacterial
cell)
Growth of Colonies
Bacterial Colony •
Result of one bacterium being isolated from –
others during “streaking procedure”
That bacterium grows in numbers exponentially –
Many bacteria have a generation time of 20 –
minutes
272 organisms in one colony after 24 hours! –
Mixed Culture of Soil Organisms
Containing Bacillus anthracis
Colony “Picking”
Sterile needle or loop •
is touched to surface
of colony and
transferred to fresh,
sterile media
Incubation for another •
24 hours
Pure Culture of Francisella tularensis
Colonies After 72 hours Growth
Pure Culture of Yersinia pestis
Colonies on Blood Agar After 48 hours of
Growth
Confirmation
Now we have a pure culture of bacteria •
Testing is now done to confirm the •
identification of the bacteria culture
Stains –
Biochemical tests –
Serological tests (using known antibodies) –
Molecular tests (nucleic acid probes) –
Yersinia pestis
Gram stain
Examples of Biochemical Tests
Left: API 50 Test
Above: Antimicrobial
Sensitivity Test
Case Study
Patient arrives in emergency room with fever •
(temperature greater than 100 degrees
F). The fever is accompanied by chills or
night sweats.
Flu-like symptoms. •
Non-productive cough, chest discomfort, •
shortness of breath, fatigue, muscle
aches
Patient Admitted to Hospital
Blood cultures ordered •
Blood drawn and immediately placed in blood •
culture bottles
Molecular Tests
Biotechnology has given diagnostic •
laboratories very powerful tools
for rapid detection and identification of human –
pathogens
for strain typing for epidemiological investigations –
The Flip Side!
Biotechnology companies attract recent college •
graduates
Majors in biology and allied fields –
Salaries usually higher than clinical or government public –
health labs offer
Appeal to public service only goes so far! –
Result: public health and clinical laboratories have •
trouble recruiting and retaining laboratory
personnel.
How to identify unknown specimen ????
Labs can grow, isolate and identify most routinely
encountered bacteria within 48h of sampling
The method microbiologist use fall into three
categories:
Phenotypic: morphology ( micro and
macroscopic)
Immunological: serological analysis
Genotypic: genetic techniques
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Phenotypic Methods
‘Old fashioned’ methods via biochemical, •
serological and morphological are still used to
identify many microorganisms.
Phenotypic methods
Microscopic morphology include a •
combination of shape, size, gram stain, acid
fat reaction, special structures e.g:
endospores, granule and capsule can be used
to give an initial presumptive identification
Phenotypic methods
• Macroscopic morphology are traits that can be
accessed with the naked eye e. g appearance of
colony including texture, shape, pigment, speed
of growth and growth pattern in broth.
• Physiology/ biochemical characteristic are
traditional mainstay of bacterial identification
• These include enzymes (catalase, oxidase,
decarboxylase), fermentation of sugars, capacity
to digest or metabolize complex polymers and
sensitivity to drugs can be used in identification
Immunological Methods
• Immunological Methods involves the
interaction of a microbial antigen with
antibody (produced by host immune system)
• Testing for microbial antigen or the production
of antibodies is often easier than test for the
microbe itself.
• Lab kits based on this technique is available
for the identification of many micoorganisms.
Genotypic Methods
• Genotypic methods involve examing the genetic
material of the organism and has revolutionized
bacterial identification and classification.
• Genotypic methods include PCR (RT- PCR, RAPDPCR, use of nucleic acid probe, PFLP and plasmid
fingerprinting
• Increasingly genotypic technique are becoming
the sole means of identifying many
microorganisms because of its speed and
accuracy.
Calssification of Microorganisms
Phenotype: physical appearance •
Genotype: Genes by molecular techniques •