Specimen Collection and Transport
The most important aspects of microbiological testing are
collection of the right specimen and transport of the
specimen to lab in a manner that insure the reliability of the
diagnostic procedure (e.g., culture, microscopy and antigen
or antibody tests).
As a general guideline for all specimens the following consideration should be
kept in mind:
Specimens should always be considered infectious. Therefore, gloves should always be
worn when handling specimens.
Specimens should be collected from the areas where organisms are present. For
example, diagnosis of a lower respiratory tract infection requires collection of material
from that site (e.g., sputum) and not from the mouth (e.g., saliva)
The quantity of specimen collected must be sufficient to ensure that all requested tests
(culture, microscopy, antigen tests ,nucleic acid probes and amplification) can be
performed properly.
Transport of specimens should maintain the viability of etiologic agent (if culture is
performed) and prevent overgrowth with contaminating organisms.
Specimens should always be transported in a leak-proof container inserted in a leakproof plastic bag.
for shipement of speciemen or isolates, refer to the International Air Transport
Association (IATA) Dangerous Goods Regulation
(http://www.IATA.org/dangerousgoods/index), the U.S. Department of Transportation
(http://hazmat.dot.gov/rules.htm) and the international Civil Aviation Organization (ICAO).
Each specimen type requires specific packing and handling procedures.
The Classification and
Identification of Bacteria of
Medical Importance
Gram negative cell wall
Gram positive cell wall
Gram stain
One of the most important
techniques in bacteriology is the
Gram stain, first described by
Hans-Christian Gram in 1884.
It allows the differentiation of
bacteria into two groups: Grampositive bacteria can retain a
complex made between crystalviolet and iodine when
decolourised with acetone,
whereas Gram-negative
bacteria are decolourised on
treatment with acetone.
the Gram stain enables the shape of bacterial cells to be observed
easily. Bacteria fall into two major classes: rod shaped bacteria are
known as 'bacilli' (singular: 'bacillus') and round or roundish bacteria are
known as 'cocci' (singular: 'coccus').
This property reveals fundamental differences in the cell envelope between the two groups. Gram-positive bacteria have many layers of peptidoglycan in
their cell wall; Gram-negative bacteria have only one or two peptidoglycan layers but, additionally, they have an outer membrane. These differences have
important consequences. For example, certain antibiotics cannot penetrate the outer membrane of Gram-negative bacteria, which are intrinsically resistant
to these drugs as a consequence.
Micrococcus luteus
Staphylococcus aureus
Bacillus subtilis
E. coli
Serratia marcescens
Performance standards for stains
Control organism/
material
ATCC No
Expected result
Ziehl-Neelsen
Mycobacterium sp.
Esch. coli
25177
25922
Pink red bacilli
Blue bacilli
Acridine orange
Esch. coli
Staph.aurues
25922
25923
Fluorescent
bacilli/cocci
Giemsa
Thin film blood smear
Gram
Esch. coli
Staph.aureus
Iodine solution
Formalin treated stool
specimen with cysts
Visible cyst nuclei
Spores
Bacillus species
Spores stain one colour and
bacillus stains with
counterstain
Stain
Distinct staining of WBCs
and RBCs
25922
25923
Gram -ve bacilli
Gram +ve cocci
Quality control of stains need to be performed on weekly basis and also as and when a new
lot of reagents for staining are procured
Staphylococcus aureus, and
shows Gram-positive cocci in grapelike clusters.
There are two major groups of Gram-positive cocci that are of medical importance: the
staphylococci and the streptococci . When viewed microscopically, staphylococci
appear in clumps, like bunches of grapes. Staphule is Greek for grapes. Streptococci
form chains, and are named after streptos, the Greek word for twisted. These groups
of bacteria can be distinguished because staphylococci produce an enzyme, catalase
and streptococci do not. Catalase causes the conversion of hydrogen peroxide to
water with the concomitant release of oxygen gas, seen as bubbles in the reaction
tube. The catalase test is a more reliable test to differentiate staphylococci from
streptococci than microscopic observation.
Streptococcus pyogenes and shows Gram-positive
cocci again, this time in chains.
Streptococci are classified according to their ability to break down blood in fresh blood
agar plates.
Some streptococci have no effect on blood. These are the non-haemolytic
streptococci (see below).
The α-haemolytic streptococci cause partial breakdown of blood, and their colonies
are surrounded by a greenish halo. The green pigment is thought to comprise the
metabolic degradation products of haem. Because of the colour of halo that surrounds ahaemolytic streptococci, they are often referred to as "viridans" streptococci (viridis is
Latin for green).
There is one α -haemolytic streptococcus that must be differentiated from the others.
This is Streptococcus pneumoniae. This is the cause of pneumococcal pneumonia
and meningitis, as well as less serious infections. Streptococcus pneumoniae is
sensitive to optochin, an antimicrobial agent. All other viridans streptococci are resistant
to optochin .
Streptococcus Gram stain
Greening Streptococcus In Blood Agar
The ß-haemolytic streptococci cause the complete breakdown of blood in fresh
blood agar plates. The colonies are surrounded by haloes that are completely
clear. Clinically, the most important of the b-haemolytic streptococci is
Streptococcus pyogenes. This belongs to the "Lancefield Group A" based upon
its antigenic structure. Streptococcus pyogenes may be differentiated from other
b-haemolytic streptococci on the basis of its sensitivity to the antibiotic bacitracin.
The most important of the non-haemolytic streptococci are the enterococci such
as Enterococcus faecalis and Enterococcus faecium. Until the early 1990's
these bacteria were classified in the genus Streptococcus but molecular biological
techniques have shown that they are sufficiently distant from other streptococci to
warrant being placed in their own genus. As their names imply, these bacteria can
be found in the gut, and can grow in the presence of bile salts.
•
Beta-Hemolytic Streptoccus; group of bacteria, most common of which is
Streptococcus pyogenes, which may cause Strep Throat, Scarlet Fever,
Glomerulonephritis, or Rheumatic Fever. Hemolysis on blood agar. LM
Gram-positive Bacilli
The genus Bacillus
The Gram-positive rods can be divided according to their ability or otherwise to
produce spores.
Spores of Gram-positive rods are highly resistant structures that may add
considerably to their pathogenic capacity.
Sporing Gram-positive rods that are confined to the (somewhat confusingly
named) genus Bacillus.
Important members of this genus include Bacillus anthracis the cause of
anthrax, and Bacillus cereus a cause of food poisoning. (Ceres was a Roman
goddess of the harvest).
The genus Bacillus also has members that produce clinically useful antibiotics,
like Bacillus polymyxa, the source of polymyxin.
The image on the left is of Clostridium perfringens, stained
from a clinical specimen using the Gram staining protocol.
Spores can be seen as areas with no stain in a few of the dark
fat rods of this bacterium. In culture, it is very difficult to
persuade this bacterium to form spores but they are seen in
clinical specimens. The spores are located towards the centre
of the bacillus in this species and they do not cause the
bacilliary body to swell.
In contrast, the spores of Clostridium tetani are
located at the end of the cell and are wider than
the bacilliary body. Sporing bacteria of Clostridium
tetani have a characteristic 'drumstick'
appearance, as seen in the image on the right.
This is a spore stain made from a pure culture of
these bacteria.
Clostridium tetani Bacteria are gram-postive rods or bacilli with
terminal spores that cause tetanus in humans, spore
Bacillus of Anthracis (Gram Stain)
Other Gram-positive bacilli
The motility of the non-sporing Gram-positive rods is an important attribute in
distinguishing coryneform bacteria and lactobacilli from listeria.
Listeria monocytogenes is an important human pathogen, and it is capable of a
characteristic tumbling motility seen at 25 degrees C but not at 37 degrees C.
Lactobacilli appear microscopically as long, slender rods that often grow in chains.
They may appear "Gram-variable" with some parts of the cell appearing blue-black
and other portions looking red. They tend to make their immediate environment too
acid for other bacteria to tolerate. Some lactobacilli are important members of the
vaginal commensal flora of women of child-bearing age. These are sometimes
referred to as Döderlein bacilli. The lactobacilli are catalase-negative, and can thus
be distinguished from the coryneform bacteria that do produce catalase.
The most infamous of the coryneform bacteria is Corynebacterium diphtheriae ,
toxigenic strains of which cause diphtheria. This gives the coryneform bacteria their
alternative name - diphtheroids. They appear somewhat irregular in shape, and tend
to cluster in Gram films. Some microbiologists think that this gives them the
microscopic appearance of Chinese letters.
Propionibacteria are coryneforms that cannot grow in the presence of air. A notable
example is Propionibacterium acnes, associated with acne.
Gram stain of a Corynebacterium seen on a skin swab
Gram stain of Corynebacterium spp.
demonstrating "Chinese letters" formations
Small, nonmotile, irregularly staining pleomorphic Gram-positive
rods with club-shaped swelled ends but no spores; may be
straight or slightly curved
C. diphtheriae and related organisms are collectively termed
coryneforms or diphtheroids
Corynebacteria possess capsular (K) and somatic antigens (O)
The top image is of a group of
corynebacteria stained using
Gram's method and clustered in
their characteristic shape. Some
bacteriologists consider these to
resemble Chinese pictograms. An
idealsied version is shown in the
lower image
Pasteurella (Francisella) tularensis) Bacteria, the gram-negative
coccobacillus pathogen that causes Tularemia or Rabbit Fever. LM X500.
Description: Gram Stain Smears of the Agents of Anthrax (Bacillus anthracis), Plague
(Yersinia pestis), and Tularemia (Francisella tularensis), Demonstrating Comparative
Morphology, Size, and Staining Characteristics (Dennis et al., 2001).
Tularemia, also known as rabbit fever or deer-fly fever, is a zoonotic disease caused by
the gram-negative intracellular pathogen Francisella tularensis (Farlow et al., 2005
Francisella tularensis Growth at 72 Hours After Inoculation
These Francisella tularensis colonies show characteristic
opalescence on cysteine heart agar with sheep blood (cultured
at 37 C for 72 hours). Note: On cysteine heart agar, F tularensis
colonies are characteristically opalescent and do not discolor the
medium (Dennis et al., 2001).
•
F. tularensis grows best in cysteine-enriched broth and
thioglycollate broth. It grows best on cysteine heart blood agar,
sheep blood agar, and on cysteine-supplemented agar such as
buffered charcoal-yeast agar, Thayer-Martin agar, and
chocolate agar. Selective agar may be useful when culturing
materials from nonsterile sites, such as sputum. Optimal
Temperature: 37 C , (Dennis et al., 2001).
Mycobacteria
The mycobacteria are a group of bacteria that are classified with other
Gram-positive bacteria on the basis of their cellular architecture, but they
possess a very waxy cell wall, and they rarely stain using conventional
protocols such as the Gram stain. They require special staining techniques in
order to be observed easily under the microscope. In the Ziehl Neelsen
technique, a strong solution of carbol fuchsin is applied to the microscope
slide which is then heated and held near boiling point for at least five minutes.
This is to allow the stain to penetrate the mycobacterial cells. Following this
treatment the stain will remain in the cell, even when the slide is treated with a
mixture of inorganic acid and alcohol. To visualise any other material on the
slide, methylene blue is applied as a counterstain. Because mycobacteria
resist decolourisation with acids and alcohol they are sometimes called acid
alcohol-fast bacilli. Important examples include Mycobacterium
tuberculosis and Mycobacterium leprae . The former causes tuberculosis,
and the latter is the cause of leprosy. It takes 6-8 weeks to grow
Mycobacterium tuberculosis in artificial culture, and as yet Mycobacterium
leprae cannot be grown in artificial culture.
Mycobacterium tuberculosis Bacteria in human sputum. LM X1600
Mycobacterium tuberculosis in sputum, stained by the Ziehl Neelsen technique
The red rods visible in the image above are cells of Mycobacterium
tuberculosis seen in a film made from a sputum sample. They have retained the
strong carbol fuchsin dye that has penetrated the cells following heating of the
slide to steam heat, even though the film has been decolourised with a mixture of
acid and alcohol. Because of this property, mycobacteria are referred to as acid
alcohol-fast. The background material has been counterstained with methylene
blue.
Gram-negative cocci
Medically, the most important of the Gram-negative cocci belong to the genus
Neisseria.
Neisseria meningitidis is an important cause of bacterial meningitis, and
Neisseria gonorrhoeae causes gonorrhoea. Members of the genus Neisseria
are most often seen in pairs, and are hence sometimes referred to as diplococci.
They are very vulnerable to drying, and can only be cultivated in an atmosphere
where the concentration of carbon dioxide is greater than that found in air. In the
laboratory, carbon dioxide incubators are used that maintain a moist environment
with a carbon dioxide concentration of 5-10%
Gram-negative cocci
Neisseria gonorrhoeae in pus from a urethral discharge
This bacterium is typically found in pairs, known as diplococci. Here the
diplococcal cells are seen within a polymorphonuclear leukocyte seen in pus from
a urethral discharge. This is sufficient information to make a provisional diagnosis
of gonorrhoea, which will be confirmed following a positive culture result,
Gram-negative bacilli
Enterobacteriaceae
This image shows the typical appearance of
Escherichia coli when stained using the Gram
staining method
The Enterobacteriaceae are a large family of medically important Gram-negative
bacilli. They can grow in the presence or absence of oxygen, and are frequently
found in the guts of humans and other animals, and hence their name. They are
differentiated from one another largely on the basis of their metabolic behaviour and
on their antigenic structure. Some, like Escherichia coli and members of the genus
Klebsiella can ferment lactose to produce acid, whereas others including
salmonellas, shigellas and proteeae cannot and are thus known as non-lactose
fermenters (NLF's). There are almost 2,000 different varieties of salmonella
recognised on the basis of differences in their surface antigens. Members of the
genus Proteus are so highly motile that a single colony can grow to swarm over the
entire surface of a Petri dish after overnight incubation. This is how the genus
acquired its name, from Proteus, the Greek sea god who had the miraculous ability
to change his shape. The family Enterobacteriaceae include Yersinia pestis, the
cause of plague, Salmonella typhi, the cause of typhoid, Shigella dysenteriae, the
cause of bacilliary dysentery, and Salmonella enteritidis implicated in many cases
of food poisoning.
Mixed bacterial colonies on MacConkey agar, Escherichia
coli (red) and Salmonella typhimurium (white).
Some bacteria are enclosed within a capsule. This protects the bacterium, even within
phagocytes, helping to prevent the cell from being killed.
Encapsulated bacteria grow as 'smooth' colonies, whereas colonies of bacteria that have lost
their capsules appear rough.
Rough colonies do not generally cause disease. Encapsulated bacteria do not succumb to
intracellular killing as easily as bacteria that lack capsules.
Cultures of Salmonella typhimurium on tryptone glucose agar (left)
and on MacConkey agar (right).
Salmonella typhosa: gram negative, brightfield. LM X500
Conventional Biochemical tubes for Shigella spp
Conventional Biochemical tubes for Salmonella spp
TABLE 1: BIOCHEMICAL IDENTIFICATION OF ENTERIC Pathogens
K
Shigella dysenteriae
K
Shigella flexneri
K
Shigella boydii
Shigella sonnei
Aeromonas Spp.
0%
K
1%
Other Salmonella spp.
8%
K
Oxidase
Salmonella pullorum
Urease
K
NaCl
U
Butt
Salmonella gallinarum
LIA
Slant
K
O
Ornithine
S. paratyphi A
+
+
-
-
K
K
-
-
-
+
-
+
K
A
-
-
A
V
-
-
V
K
V
-
-
A
V
-
-
V
K
V
-
-
+
+
-
+
K
K
-
-
A
A
-
-
V
-
K
A
-
-
-
-
V
-
K
A
-
-
K
A
-
-
V
-
K
A
-
-
K
A
-
-
-
+
K
A
-
-
A
-
+
V
-
K
A
-
+
-
+
+
K
@
A
Indole
K
I
Motility
S. typhi
M
H2S
butt
Slant
Identification
A
I
AG
AG
*
Plesiomonas spp.
K
A
-
+
+
+
K
K
-
+
-
+
+
Vibrio cholerae
K
A
-
+
+
+
K
K
-
+
-
+
+
V. parahaemolyticus
K
A
-
+
+
+
K
K
-
+
+
+
-
V. fluvialis
K
A
-
+
V
-
K
A
-
+
-
+
-
Symbols: KIA: Kligler Iron Agar, MIO: Motility Indole Ornithine Agar, LIA: Lysine Iron Agar, K: alkaline, A:
acid, G: gas, S: slant, B: butt, Ure: urease, Oxid: oxidase, * Some strains produce gas. @ Some
strains ferment lactose (KIA=A/A)
according to Farmer and Kelly, (1991)
Pseudomonads
Members of the family Enterobacteriaceae do not elaborate the enzyme complex
known as "oxidase", whereas many Gram-negative bacteria do. Pseudomonas
aeruginosa is an oxidase-positive Gram-negative bacillus that is an obligate aerobe.
It cannot be grown in the absence of oxygen. It is responsible for wound infections,
and the bacteria in this species produce a soluble pigment.
Culture of Pseudomonas aeruginosa Bacteria on
blood agar.
Curved Gram-negative rods
•
•
The vibrios and campylobacters are Gram-negative rods that appear
curved or spiral in shape. These bacteria are commonly found in natural
waters, both fresh-water and marine. Vibrio cholerae causes cholera, a
waterborne infection. Campylobacters have only been recognised as
human pathogens since the late 1970's, although they have been long
considered to be animal pathogens. Campylobacters are now responsible
for more cases of bacterial enteritis annually than salmonellas.
The bacterium Helicobacter pylori, found associated with stomach ulcers,
is a good example of a bacterium that has undergone a radical change in
taxonomy. When they were first observed, they could not be cultured and
were called "campylobacter-like organisms". Conditions were then
discovered that allowed these bacteria to be grown in artificial culture, and
they were first called Campylobacter pyloridis. This was then found to be
linguistically incorrect, and the name was corrected to Campylobacter pylori.
Later, molecular studies showed that it was not very closely related to the
other campylobacters, and they then became the type species of a new
genus, Helicobacter. This entire process of taxonomic change took less
than five years.
Campylobacter Species
speciation chart
Skirrow Campy plate
primary plate
42 degrees C
Microaerophilic
Oxidase Test
Positive
Oxidase Test
negative
Gram Stain
Gram negative curved rods
Not Campylobacter spp
Hippurate Hydrolysis
Positive
Negative
C.jejuni
Indoxyl Acetate Hydrolysis
positive
C.coli
positive
negative
Catalase test
Campylobacter
species
negativ
C.upsaliensis
Other Gram-negative bacilli
•
•
•
Some Gram-negative bacilli appear so short that they resemble cocci in the light
microscope. Because of this they are sometimes called cocco-bacilli. These include
members of the genus Moraxella, related to the neisserias, and also members of the
genus Acinetobacter. Members of this genus are increasingly associated with
hospital-acquired infection.
Some Gram-negative bacteria are very fastidious (fussy) in their nutritional
requirements. Members of the recently recognised genus Legionella, some of which
cause atypical pneumonias like Legionnaires' disease, require higher levels of iron
and cysteine than are usually present in bacteriological media, and they grow best in
media that incorporate activated charcoal to adsorb their toxic metabolic products.
Similarly bacteria of the genus Bordetella also generate toxic metabolic products that
inhibit their own artificial culture. These bacteria also grow best on media that contain
activated charcoal. Bordetella pertussis, the cause of whooping cough, is an
important member of this genus. At one time, species of the genus Bordetella were
classified in the genus Haemophilus, but they were re-classified. This is partly
because they require neither the X- nor the V-factor for growth such as required by
members of the genus Haemophilus. The X-factor has now been identified as haem,
and the V-factor is nicotinamide adenine dinucleotide or NAD. Haemophilus
influenzae requires both X- and V-factors for growth whereas Haemophilus
parainfluenzae requires just the V-factor to support its growth, since it can elaborate
its own supply of haem.
The most important group of obligately anaerobic Gram-negative bacilli are the
bacteroides. This is a heterogeneous group that form part of the human commensal
flora, and that are also implicated in anaerobic infections. The taxonomy of the
anaerobic Gram-negative rods is currently undergoing radical revision.
X and V dependence in Haemophilus influenzae
The pathogenic Haemophilus influenzae can be differentiated from its non-pathogenic relative,
Haemophilus parainfluenzae, by its dependence on an external supply of two growth factors, known as 'X'
(haem) and 'V' (NAD).
Haemophilus influenzae requires both, while Haemophilus parainfluenzae requires only the 'V' factor, since it
can make its own haem.
In the image above, the bacteria can only grow around the disc containing both 'X' and 'V' factors, indicating
that it is the pathogen, Haemophilus influenzae. X and V dependence may also be demonstrated by the
phenomenon of 'satellitism'. The second image above illustrates this. A lawn of test bacteria is plated onto a
fresh blood agar plate. This provides a supply of haem. An inoculum of Staphylococcus aureus, which can
provide NAD, is placed on the plate and the culture is incubated. Haemophili can be seen growing larger near
to the staphylococcal colony, where the supply of NAD is greatest. Colonies of haemophili growing further
from the staphylococcal colony are proportionately smaller, as the supply of NAD from the staphylococcus
diminishes. This test is good for detecting haemophili, but cannot distinguish the pathogenic Haemophilus
influenzae from Haemophilus parainfluenzae.
Haemophilus influenzae
•
Haemophilus influenzae Bacteria, a gram-negative rod that causes Bacterial
.
Meningitis. LM X500
(Haemophilus influenzae) and (Staphylococcus) satellite test
(Haemophilus influenzae) and
(Staphylococcus aureus) on blood agar,
culture
(Haemophilus influenzae) and
(Staphylococcus) satellite test
Quality control procedures for commonly used tests
Procedure/
Test
Control organism
Expected
result
Expected reaction
Catalase
Staph aureus
Streptococcus species
+
–
Bubbling reaction
No bubbling
Coagulase
Staph aureus
+
Clot formation in 4 hours
No clot
Staph epidermidis
–
Indole
Esch coli
Enterobacter aerogenes
+
–
Red ring at surface
Yellow ring at surface
Methyl red
Esch coli
Ent aerogenes
+
–
Instant red colour
No colour change
Oxidase
P. aeruginosa
+
Purple colour in 20 seconds
No colour in 20 seconds
Esch. coli
–
Voges
Proskauer
Enterobacter aerogenes
Esch. coli
+
–
Red colour
No colour change
Bacitracin disc
Streptococcus group A
Enterobacter faecalis
+
–
Zone of inhibition
No zone of inhibition
Optochin disc
Strept. Pneumoniae
Strept. viridans
+
–
Zone of inhibition
No zone of inhibition
ONPG disc
Esch. Coli
Proteus vulgaris
+
–
Yellow colour
No change in colour
Oxidase disc
P aeruginosa
+
Purple colour in 30 seconds
No change in colour
Esch. coli
–
Quality control procedures
• It is also essential to undertake quality control
procedures at regular intervals. These should be
performed:
•
With each new batch of reagents
•
With each new vial of reagent
•
Daily for catalse, oxidase, and coagulase
•
Weekly for bacitracin, optochin and ONPG
• A test procedure not giving anticipated results
with the control organisms should not be used till
such time that remedial steps have been taken
to correct the
Basic sets of drugs for routine susceptibility tests
Set 1
Set 2
Staphylococcus
Benzylpenicillin
Oxacillin
Erythromycin
Tetracycline
Chloramphenicol
Gentamicin
Amikacin
Co-trimoxazole
Clindamycin
-
Intestinal
Ampicillin
Chloramphenicol
Co-trimoxazole
Nalidixic acid
Tetracycline
Norfloxacin
Enterobacteriaceae
Urinary
Sulfonamide
rimethoprim
Co-trimoxazole
Ampicillin
Nitrofurantoin
Nalidixic acid
Tetracycline
Norfloxacin
Chloramphenicol
Gentamicin
Blood and tissues
Ampicillin
Chloramphenicol
Co-trimoxazole
Tetracycline
Cefalotin
Gentamicin
Cefuroxime
Ceftriaxone
Ciprofloxacin
Piperacillin
Amikacin
Piperacillin
Gentamicin
Tobramycin
Amikacin
Pseudomonas aeruginosa
Factors influencing zone size in antibiotic susceptibility testing
Factor
Influence
Inoculum density
Larger zones with light inoculum and vice versa
Timing of disc application
If after application of disc, the plate is kept for longer time at room
temperature, small zones may form
Temperature of incubation
Larger zones are seen with temperatures < 35oC
Incubation time
Ideal 16-18 hours; less time does not give reliable results
Size of the plate
Smaller plates accommodate less number of discs
Depth of the agar medium
Thin media yield excessively large inhibition zones and vice versa
Proper spacing of the discs
Avoids overlapping of zones
Potency of antibiotic discs
Deterioration in contents leads to reduced size
Composition of medium
Affects rate of growth, diffusion of antibiotics and activity of antibiotics
Acidic pH of medium
Tetracycline, novobiocin, methicillin zones are larger
Alkaline pH of medium
Aminoglycosides, erythromycin zones are larger
Incubation in the presence of CO2
Increases zone size of tetracycline and methicillin
Addition of thymidine to medium
Decreases activity of trimethoprim
Addition of defibrinated blood
Decreases activity of sulfonamides
On chocolate agar, decreased activity of
Sulfonamides, trimethoprim, aminoglycosides
Reading of zones
Subjective errors in determining the clear edge
Chelating agents such
magnesium and iron
as
cal-cium,
Decreases diffusion of tetracycline and gentamicin
Need for quality control in susceptibility test
• The final result of a disc diffusion test is influenced by a large number of
variables. Some of the factors, such as the inoculum density and the
incubation temperature, are easy to control, but a laboratory rarely
knows the exact composition of a commercial medium or the batch-tobatch variations in its quality, and it cannot take for granted the
antimicrobial content of the discs. The results of the test must, therefore,
be monitored constantly by a quality control programme which should be
considered part of the procedure itself.
• The precision and accuracy of the test are controlled by the parallel use
of a set of control strains, with known susceptibility to the antimicrobial
agents. These quality control strains are tested using exactly the same
procedure as for the test organisms. The zone sizes shown by the
control organisms should fall within the range of diameters given in
Table 11.4. When results regularly fall outside this range, they should be
regarded as evidence that a technical error has been introduced into the
test, or that the reagents are at fault. Each reagent and each step in the
test should then be investigated until the cause of the error has been
found and eliminated
Quality Control – Susceptibility of Control Strains*
Diameter of zone of inhibition (mm )
Antibiotic
Disc potency
Staph.aureus
(ATCC 25923)
Esch.coli
(ATCC 25922)
P.aeruginosa
(ATCC 27853)
Amikacin
30 mg
20-26
19-26
18-26
Ampicillin
10 mg
27-35
16-22
–
Ceftriaxone
30 mg
22-28
29-35
17-23
Cephalothin
30 mg
29-37
15-21
–
Chloramphenicol
30 mg
19-26
21-27
–
Ciprofloxacin
5 mg
22-30
30-40
25-33
Clindamycin
2 mg
24-30
–
–
Erythromicin
15 mg
22-30
–
–
Gentamicin
10 mg
19-27
19-26
16-21
Nalidixic acid
30 mg
–
22-28
–
Nitrofurantoin
300 mg
18-22
20-25
–
Norfloxacin
10 mg
17-28
28-35
–
Oxacillin
1 mg
18-24
–
–
Penicillin G
10 units
26-37
–
–
Piperacillin
100 mg
–
24-30
25-33
Tetracycline
30 mg
19-28
18-25
–
Tobramycin
10 mg
19-29
18-26
19-25
*NCCLS: 1995
Trimethoprim
5 mg
19-26
21-28
–
Trimethoprimsulfamethoxazole
1.25/23.75
24-32
24-32
–
Troubleshooting guide for disc diffusion test in antibiotic susceptibility testing
Aberrant results
Probable cause
Tetracycline zone too small
pH of medium too low
Aminoglycoside zone too small
pH of medium too high
Aminoglycoside zone too large
Ca2+ and/or Mg2+ level too high in medium
Ca2+ and/or Mg2+ level too low in medium
Too large zone on control plates
Inoculum too light
Nutritionally poor medium
Slow growing organisms (not seen with controls)
Improper medium depth (too thin)
Zone universally too small on control plates
Inoculum too heavy
Methicillin zone indeterminant in disc test
Methicillin degraded by strong b lactamase producing staphylococci
Carbenicillin zone disappears with Pseudomonas
control
Resistant mutant has been selected for testing
Single disc result above or below control limits
Error in reading, fuzzy zone edge, transcription error, bad disc
Disc may not be pressed firmly onto agar surface
Colonies within zone of inhibition
Mixed culture
Resistant mutants within zone
Zones overlap
Discs too close together
Zones indistinct
Poorly streaked plates
Zone within zone phenomenon
Swarming Proteus species
Feather edge of zones around penicillin or ampicillin discs usually with
b lactamase negative strains of Staph.aureus
PCR
Gram negative cell wall
Gram positive cell wall
Back to FRET
Fluorescence Resonance Energy Transfer
Reporter-Quencher probe system that allows the detection, and
quantifying, of nucleotide amplification in real time.
R
R
Q
e-
Q
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TaqMan
Bacteria
The bacterial cell lacks a membrane-bound nucleus. Because of
this, bacteria are described as prokaryotes, "pro-" meaning "before"
and "-karyon" from the Greek word for a "nucleus".
There are three basic shapes that bacterial cells adopt. They are
either round, rod shaped or spiral.
Round bacteria are referred to as cocci (singular: coccus),
and rod shaped bacteria are known as bacilli (singular: bacillus).
The term 'bacillus' meaning a rod-shaped bacterium should NOT be
confused with the genus of bacteria known as 'Bacillus'.
Staphylococci
Diplococcal cells of
Streptococcus pnuemoniae
Streptococci
Sporing cells of
Clostridium tetani
Note spores do not stain and in this case
cause the bacilli to swell
Sporing cells of
Bacillus anthracis
Note spores do not cause the bacilli to swell in this
species
Spiral cells of
Treponema pallidum
This bacterium causes syphilis and is so slender that it
cannot be seen using conventional light microscopy.
It is most easily visualized using dark-ground microscopy.
Irregular cells of
Corynebacterium diphtheriae
Various shaped cells of
Haemophilus influenzae
Curved rods of Vibrio cholerae
• Strains of Streptococcus pnuemoniae that lack
capsules do not cause disease. All the bacteria
that cause meningitis are encapsulated.
• Suspending bacteria in India ink is an easy way
of demonstrating capsules. Ink particles cannot
penetrate the capsular material and
encapsulated cells appear to have a halo around
them. This is the Quellung reaction.
• In the 'Quellung' reaction, bacterial cells are
resuspended in antiserum that carries antibodies
raised against the capsule. This causes the
capsule to swell, and this can be easily
visualised by suspension in India Ink. The ink
particles cannot penetrate the capsule, which
this appears as a halo around the bacterial cells.
The Quellung reaction
Endospores (or simply spores)
•
A few species of bacteria have the ability to produce highly resistant
structures known as endospores (or simply spores). These resist a range of
hazardous environments, and protect against heat, radiation, and
desiccation.
•
Endospores form within (hence endo-) special vegetative cells known as
sporangia (singular sporangium).
•
Diseases caused by sporing bacteria include botulism (Clostridium
botulinum),
Ggas gangrene (Clostridium perfringens),
Ttetanus (Clostridium tetani)
Acute food poisoning (Clostridium perfringens, again)
All these bacteria are 'anaerobic'.
The aerobic sporing bacteria can also cause disease. Anthrax is caused by
Bacillus anthracis. Bacillus cereus causes two types of food poisoning.
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Microfungi
Microfungi
All fungi are eukaryotic.
Most possess a cell wall made of chitin: a polymer of N-acetyl glucosamine that is found in the
cell walls of the majority of fungi. It is also a major component of the exoskeleton of arthropods
such as insects. The cell walls of plants are made of cellulose whereas the walls of
fungal cells are made of chitin and other polymers. Chitin is also the material found
in insect shells.
The dreaded•).
black mould in the bathroom of the student flat? This is most likely to be a fungus
of the genus Cladosporium.
Who has not seen Penicillium spp. growing on stale bread?
These are all moulds: fungi that grow in mats of tiny filaments known as hyphae (singular:
hypha, Greek for a thread) or mycelia (singular: mycelium, from the Greek mukes, meaning a
mushroom). These may or may not be subdivided into separate compartments by cross walls
known as septa (singular: septum).
Aseptate and septate mycelia
Moulds are multicellular organisms.
There are, however, unicellular fungi: the yeasts.
Most familiar of the yeasts is Saccharomyces cerevisiae. Depending upon your
viewpoint, this is baker's yeast, used in the production of leavened bread, or
brewer's yeast, used in alcohol production.
Yeasts grow and multiply by budding daughter cells off from a mother cell.
Budding Yeasts
Fungi can multiply either sexually or asexually.
In classifying fungi, great weight is placed upon the
appearance and structure of sexual fruiting bodies.
Any fungus that does not exhibit a recognizable sexual
structure is difficult to classify. They are grouped in the
collection known as "fungi imperfecti". Among the most
important of fungi imperfecti are members of the genus
Penicillium. It is from these fungi that we derive penicillins.
These were the first true antibiotics and are still among the
most used antimicrobial agents world-wide.
Fungi also produce a staggering variety of spores. These may be produced
asexually or sexually and are important in the identification of moulds.
Examples of asexual spore structures produced by fungi
The umbrella is the spore-bearing
structure once it has discharged its
load
Asexual spores can be
enclosed within
specialised sacs
The umbrella is the spore-bearing structure once it has discharged its load
Examples of sexual spore structures produced by fungi
Spores within an enclosed
structure - a cleistothecium
The warty zygospore is
suspended between two
mycelia of different mating
types
Spores contained inside a more
open structure - a perithecium
Moulds cause a variety of common, superficial infections such as ringworm and
athlete's foot.
In compromised individuals they can cause much more severe infections but these
are rare.
The most common yeast infection is "thrush" caused by Candida albicans. Under
certain conditions, some yeasts have the ability to develop pseudomycelia. This
happens, for example, when the commensal form of Candida albicans causes
active thrush.
"Pseudomycelia" produced by Candida albicans
Gram-stain of vaginal smear showing Candida albicans, epithelial
cells, and many gram-negative rods
Gram-stain of vaginal smear showing Candida albicans epithelial cells and many
gram-negative rods. (1,000X oil)
Viruses
Viruses
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Viruses are obligate intracellular parasites (requires to live within a cell in its
host).
They comprise a nucleic acid core wrapped in a protein coat. Some viruses
have an envelope, made of lipid and usually derived from the cell in which they
grow.
Other viruses are naked and just have their protein coat exposed, protecting the
nucleic acid within the centre of the structure.
Specific viruses attack specific types of cells. Respiratory syncytial virus infects
only the cells of the respiratory tract, for example.
There is a special class of virus that attacks bacteria. These are the
bacteriophage.
Most viruses are simple in shape: round, rod-shaped, icosahedral, brick-shaped
or bullet-shaped
Viruses have a nucleic acid core, either DNA or RNA but not both. Retroviruses
are unusual in that the virion carries an RNA copy of the genome but upon
infection of a host cell a cDNA copy of the virus genome is made using the
enzyme reverse transcriptase
Human immunodeficiency virus
- the cause of AIDS
Around the nucleic acid core lies a protein coat, made up of units called
capsomeres. Some viruses also have an envelope derived from the host cell
membranes. The envelope may be either baggy or tight, depending upon the nature
of the virus.
•.
Rabies virus
Adenovirus
- associated with respiratory and gastrointestinal disease
A T-even bacteriophage that can infect Escherichia coli
Herpes simplex virus
(note the lipid envelope)