Chapter 3.3-Lab Diagnosis of Bacterial Infection

GENERAL BACTERIOLOGY:
LABORATORY DIAGNOSIS
OF
BACTERIAL INFECTIONS
Learning objectives
At the end of the session, the students will be able to understand:

▰ Specimen Collection
▰ Direct Detection, Staining Techniques
▰ Culture, Identification and AST
▰ Serology
▰ Molecular Methods
3
Essentials of Medical Microbiology
LABORATORY DIAGNOSIS OF
BACTERIAL INFECTIONS
Useful for the following purposes:
▰ Identification
▰ Treatment
▰ Surveillance purpose
▰ For outbreak investigation
▰ To start PEP (post-exposure prophylaxis)
▰ To initiate appropriate infection control measures
4
LABORATORY DIAGNOSIS OF
BACTERIAL INFECTIONS (Cont..)
Comprises of several steps
▰ Specimen collection
▰ Direct detection
▰ Culture
▰ Identification and Antimicrobial susceptibility test
▰ Serology
▰ Molecular methods
5
SPECIMEN
COLLECTION
6
Types of infections and various specimens
collected
Type of infections Specimens collected
Blood stream infection, sepsis, endocarditis Paired blood culture specimens

• Collected aseptically by two-step
disinfection of skin; first with alcohol
followed by chlorhexidine
• 8-10mL of blood (for adults) collected in
blood culture bottles
Infectious diseases requiring serology • Blood (2 mL/investigation)
• Collected by minimal asepsis (one-step
skin disinfection with alcohol)
• Collected in vacutainer
Diarrheal diseases Stool (mucus flakes), rectal swab
Meningitis Cerebrospinal fluid (CSF)
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collected (Cont..)
Infections of other sterile body area Sterile body fluids; e.g. pleural fluid, synovial fluid, peritoneal fluid
Skin and soft tissue infection Pus or exudate, wound swabs, aspirates from abscess and tissue bits
Anaerobic culture Aspirates, tissue specimens, blood and sterile body fluids, bone marrow
(swabs, sputum not satisfactory)
Upper respiratory tract infection Throat swab with membrane over the tonsil, nasopharyngeal swab, per-
nasal swab
Lower respiratory tract infection Sputum, endotracheal aspirate, bronchoalveolar lavage (BAL),
protected specimen brush (PSB) and lung biopsy
Pulmonary tuberculosis • Sputum- early morning and spot
• Collected in well-ventilated area
• Gastric aspirate for infants
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collected (Cont..)
Urinary tract infections Midstream urine

Suprapubic aspirated urine
Catheterized patient: collected from the
catheter tube, not from urobag
Genital specimens Urethral swab, cervical swab- for urethritis
Exudate from genital ulcers
Eye infections Conjunctival swabs
Corneal scrapings
Aqueous or vitreous fluid
Ear infections Swabs from outer ear
Aspirate from inner ear
9
General Principles
Following principles should be followed while collecting the specimen:
▰ Standard precautions
▰ Before antibiotics start
▰ Contamination with indigenous flora should be avoided
▰ Swabs - convenient but considered inferior to tissue, aspirate and body

fluids
10
General Principles (Cont..)
▰ Container: sterile, tightly sealed, leak proof, wide-mouth
▰ Labeling: All specimens - labelled with name, age, gender, treating

physician, diagnosis, etc.
▰ Rejection: Specimens contaminated or improperly labelled may be rejected
▰ Anaerobic culture - proper anaerobic collection containers with media

should be used.
11
Specimen Transport
Most specimens - transport time should not exceed two hours. However, there
are some exceptions.
▰ Immediate transport (<15 minutes) - CSF and body fluids, ocular

specimens, tissue specimens, suprapubic aspirate and bone specimen.
▰ Urine (midstream) - added with preservative (boric acid) - transported

within 2 hours.
12
Specimen Transport (Cont..)
▰ Stool culture - transported within 1 hour.
▰ Rectal swabs - up to 24 hours is acceptable.
▰ For anaerobic culture - Robertson’s cooked meat broth or any specialized

anaerobic transport system.
13
Specimen Storage before Processing
Most specimens - stored at room temperature - up to 24 hours.
There are some exceptions:
▰ Blood cultures - incubated at 37°C immediately up on receipt.
▰ Sterile body fluids - immediately plated up on receipt - incubated at 37°C.
▰ Corneal scrapping - plated at bed-side.
14
Specimen Storage before Processing
(Cont..)
▰ Stool culture - stored up to 72 hours at 4°C
▰ Urine (mid-stream and from catheter), lower respiratory tract

specimen, gastric biopsy (for Helicobacter pylori) - stored up to 24 hours
15
DIRECT
DETECTION
16
STAINING TECHNIQUES
▰ Structural details of bacteria cannot be seen under light microscope due to
lack of contrast.
▰ It is necessary to use staining methods to produce color contrast and

thereby increase the visibility.
▰ Before staining - smears are fixed - so they will not be displaced during the
staining process.
17
STAINING TECHNIQUES (Cont..)
▰ Fixation - protects the internal structures of cells.
▰ Done by two methods.
1. Heat fixation: Gentle flame heating an air-dried film of bacteria.
2. Methanol fixation—used for blood smears.
18
Common staining techniques
▰ Simple stain: Basic dyes (methylene blue or basic fuchsin) are used to
provide the color contrast, but imparts the same color to all the bacteria in a
smear.
▰ Negative staining: A drop of bacterial suspension is mixed with dyes

(India ink or nigrosin). The background gets stained black where as
unstained bacterial/yeast capsule stand out in contrast.
19
Common staining techniques (Cont..)
▰ Impregnation methods: Bacterial cells and structures are thickened by

impregnation of silver salts on their surface to make them visible, e.g. for
demonstration of bacterial flagella and spirochetes.
20
Common staining techniques (Cont..)
▰ Differential stain: Two stains are used - impart different colors to different
bacteria. Most commonly used differential stains are:
1. Gram stain: Differentiates bacteria into gram-positive and gram-
negative groups.
2. Acid-fast stain: Differentiates bacteria into acid fast and non acid-
fast groups.
3. Albert stain: Differentiates bacteria having metachromatic granules
from other bacteria that do not have them.
21
Gram Stain
Principle and procedure of Gram
▰ Originally developed by Hans staining
Christian Gram (1884).
▰ Gram stain still remains the

most widely used test in
diagnostic bacteriology.
22
Interpretation of Gram Stain
Gram staining demonstrating violet-colored
▰ Gram-positive bacteria resist gram-positive cocci in clusters and pink
colored gram-negative bacilli in scattered
decolorization and retain the color of arrangement
primary stain i.e. violet.
▰ Gram-negative bacteria are decolorized

and, therefore, take counterstain and
appear pink.
23
Principle of Gram Staining
pH theory:
▰ Cytoplasm of gram-positive bacteria - more acidic -can retain the basic dye
(e.g. crystal violet) for longer time.
▰ Iodine serves as mordant - combines with the primary stain to form a dye-
iodine complex - gets retained inside the cell
24
Principle of Gram Staining (Cont..)
Cell wall theory:
▰ Gram-positive cell wall - thick peptidoglycan layer (50–100 layers thick).
▰ Acts as permeability barrier preventing loss of crystal violet.
▰ Alcohol is thought to shrink the pores of the thick peptidoglycan.
25
Principle of Gram Staining (Cont..)
Cell wall theory (Cont..):
▰ Gram-negative cell wall - more permeable thus allowing the out flow of
crystal violet easily.
▰ Thin peptidoglycan layer in gram-negative cell wall - is not tightly cross

linked
▰ Presence of lipopolysaccharide layer in the cell wall of gram-negative

bacteria - gets disrupted easily by the decolorizer
26
Modifications of Gram Staining
Modifications Explanation
Kopeloff and Primary stain - methyl violet
Beerman’s Counter stain - basic fuchsin
Jensen’s Absolute alcohol as decolorizer and neutral red as
counter stain. Useful for meningococci and
gonococci
Brown and Brenn Used for Actinomycetes.
27
Uses of Gram Stain
▰ To differentiate bacteria into gram-positive and gram negative
▰ Identification of Gram staining from bacterial culture helps in further

identification of bacteria
▰ Initiation of empirical treatment with broad spectrum antibiotics - started

early before the culture report is available.
▰ Early presumptive identification of fastidious organisms - Haemophilus.

28
Uses of Gram Stain
▰ Gram stain gives a preliminary clue in anaerobic culture (Clostridium)
▰ Gram stain is useful for staining certain fungi such as Candida and
Cryptococcus (appear gram-positive)
▰ Helps in screening the quality of the sputum specimen before processing it

for culture.
29
Acid-fast Stain
▰ Discovered by Paul Ehrlich and subsequently modified by Ziehl and

Neelsen.
▰ Acid fastness - due to presence of mycolic acid in the cell wall.
30
Ziehl-Neelsen Technique (Hot Method)
Smear preparation and heat fixation:
▰ Smear = 2 × 3 cm in size from the yellow purulent portion of the sputum.
▰ Smear - neither be too thick nor too thin - When placed over a printed
matter, the print should be readable through the smear.
▰ The smear is air dried for 15–30 minutes and then heat fixed by passing
over the flame 3–5 times for 3–4 seconds.
31
Procedure of Ziehl-neelsen technique (Hot Method)
▰ Step 1 (primary stain) - Carbol fuchsin (1%) for 5 minutes. Intermittent

heating is done by flaming until the vapours rise.
▰ Step 2 (decolorization) - 25% sulfuric acid for 2–4 minutes.
▰ Step 3 (counter staining) - 0.1% methylene blue slide for 30 seconds.
32
Interpretation
▰ Mycobacterium tuberculosis
appears as long slender, straight or
slightly curved and beaded, red
colored acid fast bacillus.
33
Modifications of Acid-Fast Staining
▰ Cold method (Kinyoun’s method): Intermittent heating is not required.
▰ Acid-alcohol can be used as decolorizer alternatively.
▰ Malachite green can be used as counter stain.
▰ Concentration of sulfuric acid - vary depending on the acid-fastness of the

structure to be demonstrated.
34
Acid-fast organisms/structures and percentage of
sulfuric acid suitable for staining
Acid fast organisms /structures Sulphuric acid (%) needed for
decolorization
Mycobacterium tuberculosis 25%
Mycobacterium leprae 5%
Nocardia 1%
Acid fast parasites such as Cryptosporidium , 1%
Cyclospora, Cystoisosopra, Microsporidia
Bacterial spore 0.25-0.5%
35
Albert Stain
▰ Used to demonstrate - metachromatic granules of Corynebacterium

diphtheriae.
Procedure
▰ Heat fixation Smear is covered with Albert I (Albert’s stain) for 5
minutes drain the solution Albert II (iodine solution) is
added for 1 minute Slide is washed with water
36
Composition
Composition of Albert stain includes:
▰ Albert I: Comprises of toluidine blue , malachite green, glacial acetic acid ,

alcohol (95% ethanol), and distilled water
▰ Albert II: Contains iodine in potassium iodide
37
Interpretation
▰ Corynebacterium diphtheriae appears as green colored bacilli arranged in

Chinese letter or cuneiform pattern, with bluish black metachromatic
granules at polar ends.
▰ Differentiated from diphtheroids- do not show granules and are arranged in

palisade pattern.
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OTHER METHODS
OF DIRECT
DETECTION
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Antigen Detection
Various immunological methods such as latex agglutination test,
immunochromatographic test are available which detect antigens in clinical
specimens.
Examples:
▰ Detection of capsular antigen of pneumococci, meningococci, H.

influenzae in CSF specimen, etc.
40
Molecular Diagnosis
▰ Bacterial DNA or RNA can be directly detected in the clinical specimens

by various molecular methods such as polymerase chain reaction (PCR).
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CULTURE,
IDENTIFICATION AND
AST
42
CULTURE MEDIA
▰ Liquid or solid substance - contains nutrients to support the growth, and

survival of microorganisms.
43
Constituents of Culture Media
Constituent Explanation
Water and Sodium chloride

Electrolytes
Peptone Mixture of partially digested proteins - obtained from various sources - heart muscle,
casein or fibrin, or soya.
Agar Used for solidifying the culture media

Source: Prepared from - cell wall of seaweeds and available commercially in powder
form
Preparation: Agar powder is dissolved in water and subjected to sterilization by
autoclave. When the temperature of the molten agar comes down to 45°C, it is poured into
the Petri dishes and then allowed to set for 20 minutes.
Concentration:
Solid agar preparation - 1-2%
Semisolid agar- 0.5%
Solid agar to inhibit Proteus swarming- 6%
44
Constituents of Culture Media (Cont..)
Constituent Explanation
Meat extract Highly concentrated meat stock, usually made from beef.
Yeast extract (Prepared from Baker’s yeast) and malt extract (contains maltose)
Blood and serum Important components of enriched media; provide extra nutrition to fastidious
bacteria. 5–10% of sheep blood is used. Alternatively, horse, ox or human
blood can also be used.
45
Types of Culture Media
Based on consistency, culture media are grouped into:
▰ Liquid (or broth) media
▰ semisolid media
▰ solid media
46
Types of Culture Media
Based on the growth detection, culture media are classified as:
Conventional culture media: Automated culture media:

Prepared from nutrients - aqueous extract Mainly available for blood and sterile body
of meat, peptone. fluid culture.
 Simple/basal media
 Enriched media
 Enrichment broth
 Selective media
 Differential media
 Transport media
 Anaerobic media.
47
Simple/Basal Media
Contain minimum ingredients that support the growth

of non-fastidious bacteria.
▰ Peptone water: Contains peptone (1%) + NaCl
(0.5%) + water
▰ Nutrient broth: Peptone water + meat extract
(1%).
▰ Nutrient agar: Nutrient broth + 2% agar
▰ Semisolid medium: Concentration of agar -
reduced to 0.2–0.5 %. 48
Uses of Basal Media
▰ Testing the non-fastidiousness of bacteria.
▰ Serve as base for the preparation of many other media.
▰ Nutrient broth - used for studying the bacterial growth curve.
▰ Nutrient agar - preferred medium for:
 Performing the biochemical tests, such as oxidase, catalase, etc.
 Study the colony morphology
 Pigment demonstration.
49
Enriched Media
▰ Basal medium added with additional nutrients - blood, serum or egg
▰ In addition to non-fastidious organisms, support the growth of fastidious

nutritionally exacting bacteria.
50
Enriched Media (Cont..)
Blood agar –
▰ Prepared by adding 5-10% of sheep blood to the

molten nutrient agar at 450C.
▰ Tests the hemolytic property of the bacteria, which

may be either- i) partial or α (green) hemolysis and
ii) complete or β hemolysis
51
Chocolate agar:
▰ Heated blood agar, prepared by adding 5 -10%

of sheep blood to the molten nutrient agar at
70°C.
▰ More nutritious than blood agar - supports

certain highly fastidious bacteria -
Haemophilus influenzae that does not grow on 52
Loeffler’s serum slope: Contains serum - used for isolation of

Corynebacterium diphtheriae
Blood culture media: Used for isolating microorganisms from blood -

available as conventional or automated blood culture media.
53
Enrichment Broth
▰ Liquid media added with inhibitory agents which selectively allow certain
organism to grow and inhibit others.
▰ Important for isolation of pathogens from clinical specimens which also

contain normal flora (e.g. stool and sputum specimen).
54
Enrichment Broth (Cont..)
Examples :
▰ Tetrathionate broth—Used for Salmonella Typhi
▰ Gram-negative broth—Used for Shigella
▰ Selenite F broth—Used for Shigella
▰ Alkaline peptone water (APW)—Used for Vibrio cholerae.
55
Selective Media
Solid media containing inhibitory substances - inhibit the normal flora present
in the specimen and allow the pathogens to grow.
Media Used for isolation of
Lowenstein Jensen (LJ) medium Mycobacterium tuberculosis
Thiosulphate Citrate Bile salt Sucrose (TCBS) Vibrio species
DCA (Deoxycholate Citrate Agar) Salmonella and Shigella from stool
XLD (Xylose Lysine Deoxycholate) agar Salmonella and Shigella from stool
Potassium tellurite agar (PTA) Corynebacterium diphtheriae
56
Selective Media (Cont..)
Lowenstein–Jensen medium TCBS

agar
57
Selective Media (Cont..)
DCA (Deoxycholate Citrate Agar) XLD (Xylose Lysine Deoxycholate) ag
58
Transport Media
▰ Used for the transport of the clinical specimens suspected to contain

delicate organism or when delay is expected while transporting the
specimens from the site of collection to the laboratory
▰ Bacteria do not multiply in the transport media - they only remain viable.
59
Transport Media (Cont..)
Organism Transport media

Neisseria Amies medium, Stuart’s medium
Vibrio cholerae VR (Venkatraman-Ramakrishnan) medium
Autoclaved sea water
Cary Blair medium
Shigella, Salmonella Buffered glycerol saline
Cary Blair medium
60
Differential Media
▰ Differentiate between two groups of bacteria - by using an indicator.
Differential media Features

MacConkey agar
 Differential and low selective medium - used for the isolation of
enteric GNB.
 Differentiates organisms into LF (pink colonies, e.g. Escherichia
coli) and NLF or (colorless colonies, e.g. Shigella).
 Composition- Peptone, lactose, agar, neutral red (indicator) and
taurocholate
61
Differential Media (Cont..)
Differential media Features

CLED agar (cysteine lactose
 Capable of differentiating between LF and NLF.
electrolyte-deficient agar)
 Used as an alternative to combination of blood agar and
MacConkey agar, for the processing of urine specimens
62
Differential Media (Cont..)
MacConkey agar CLED agar
63
Anaerobic Culture Media
▰ Contain reducing substances which take up oxygen and create lower redox
potential - permit the growth of obligate anaerobes, such as Clostridium.
Media Features
Robertson’s cooked meat (RCM) broth  Contains chopped meat particles (beef heart), which
provide glutathione (a sulfhydryl group containing
reducing substance) and unsaturated fatty acids.
 Widely used
 Used for maintenance of stock cultures.
64
Anaerobic Culture Media (Cont..)
Other anaerobic media include:
▰ Thioglycollate broth
▰ Anaerobic blood agar
▰ BHIS agar (Brain-heart infusion agar) with supplements (vitamin K and
hemin)
▰ Neomycin blood agar
▰ Egg yolk agar
▰ Phenyl ethyl agar
▰ Bacteroides bile esculin agar (BBE agar). 65
Blood Culture Media
▰ Recovery of bacteria from blood is difficult - they are usually present in

lesser quantity and many of the blood pathogens are fastidious.
▰ Therefore, enriched media - used for isolating microorganisms from blood.
▰ Available either as conventional or automated media
66
Conventional Blood Culture Media
Two types:
▰ Monophasic medium: Contains brain–heart

infusion (BHI) broth.
▰ Biphasic medium: Liquid phase containing

BHI broth and a solid agar slope made up of
BHI agar
67
Disadvantages of conventional media
Subcultures are made manually - performed less-frequently (once a day) as it
is cumbersome.
Broth Disadvantages
Monophasic BHI broth  Subcultures are made, periodically for 1 week.
 Higher risk of contamination - due to opening of the cap of the bottle
every time when subcultures are made.
Biphasic BHI broth  Subcultures - made just by tilting the bottles so that the broth runs over
the agar slope.
 Lower risk of contamination as it obviates the opening of the cap of the
bottle
68
Automated Blood Culture Techniques
▰ Continuous automated monitoring: Blood culture bottles - periodically

monitored for the microbial growth - every 10 minutes by the instrument.
▰ Composition: Tryptic soy broth and/or brain heart infusion broth +

polymeric resin beads - adsorb and neutralize the antimicrobials present in
blood specimen.
69
Automated Blood Culture Techniques (Cont..)
▰ Specimens: Used for culture of blood, bone marrow and sterile body fluids.
▰ More sensitive: Higher yield of positive cultures from clinical specimens
▰ Rapid: Less time than conventional methods
▰ Less labor intensive: fully-automated
70
Automated Systems
BacT/ALERT 3D:
▰ Principle - colorimetric detection of

growth.
▰ When bacteria multiply - produce CO2 -

increases the pH - changes the color of a
blue-green sensor present at the bottom
of the bottle to yellow Essentials of Medical Microbiology
71
Automated Systems (Cont..)
BacT/ALERT VIRTUO (bioMerieux):
Advanced form of BacT/ALERT – has
several advantages, such as:
▰ Automatic loading and unloading of
bottles,
▰ Faster detection of growth,
▰ Determine volume of blood present in
bottle
72
Automated Systems (Cont..)
BACTEC (BD Diagnostics)
▰ Principle - fluorometric detection of growth.
▰ Uses an oxygen-sensitive fluorescent dye present in the medium.
73
Disadvantages of automated systems
▰ High cost of the instrument and culture bottles,
▰ Inability to observe the colony morphology as liquid medium is used.
74
CULTURE METHODS
75
CULTURE METHODS
▰ Involve inoculating the specimen on to appropriate culture media, followed

by incubating the culture plates in appropriate conditions.
76
Selection of Media
Specimens Recommended culture media
Pus, wound swab, aspirates, and Blood agar + MacConkey agar

tissue bits
Sterile body fluids Blood agar, plus MacConkey agar, plus chocolate agar OR
Automated blood culture bottles
Blood Blood culture bottles (Conventional or automated)
Urine Blood agar plus MacConkey agar CLED agar can be used
alternatively
Stool Selenite-F broth plus MacConkey agar plus DCA and/or XLD agar
(if cholera is suspected- add TCBS agar)
Respiratory specimens Blood agar, plus MacConkey agar, plus chocolate agar
77
Inoculation of the Specimens
▰ Carried out by bacteriological loops made up of platinum or nichrome

wire.
▰ Inoculating loop - heated in the Bunsen flame - then made cool waiting for
10 seconds.
▰ Carried out in biological safety cabinet and wearing appropriate personal

protective equipment.
78
Inoculation of the Specimens (Cont..)
A. Bacteriological loop and straight wire B. Flaming the loop (red hot)
79
Biosafety Cabinet (BSC)
▰ Enclosed, ventilated laboratory workstation -

protect the laboratory personnel while
working.
▰ Air is blown into the cabinet away from the

worker and exhausted outside through a
duct lined with HEPA filters.
80
Inoculation Methods
Two types:
▰ Methods used for inoculating clinical specimens on to the culture media
▰ Methods used for inoculating colonies on to various media for further

processing.
81
Streak Culture
▰ Most common inoculation method
▰ Used for - inoculation of the specimens on to the solid media.
▰ Used for obtaining individual isolated colonies from a mixed culture of

bacteria.
82
Streak Culture (Cont..)
Streaking: Loopful of specimen - smeared

onto solid media to form round-shaped
primary inoculum - spread over the culture
plate by streaking parallel lines to form the
secondary, tertiary inoculum and finally a
feathery tail end
83
Streak Culture (Cont..)
Intermittent heating: Loop is flamed and

cooled in between the different set of streaks
to get isolated colonies on the final streaks.
84
Liquid Culture
▰ Used for culture of specimens - blood or body fluids - inoculated by

directly adding the specimen in to the liquid medium or with the help of a
syringe or pipette.
85
Liquid Culture (Cont..)
Bacterial growth - detected by observing

turbidity in the medium. Some aerobic
bacteria form surface pellicles.
86
Uses:
▰ Blood, or body fluids culture,
▰ Automated culture for mycobacteria
▰ Water analysis
87
Advantages: Preferable for culture of:
▰ Specimens containing small quantity of bacteria
▰ Specimens containing antibiotics and other antibacterial substances - get

neutralized by dilution in the medium
▰ Large yields of bacteria are required
88
Disadvantages:
▰ Do not provide a pure culture from a mixed inoculum
▰ No visible colonies - does not given
89
Lawn or Carpet Culture
▰ Useful to carry out antimicrobial

susceptibility testing (AST) by disk
diffusion method.
▰ Uniform lawn of bacterial growth is

obtained - by either swabbing or flooding
with a bacterial broth onto the culture plate.
90
Pour Plate Technique
▰ Seldom used for quantifying the bacterial load present in the specimens -
urine or blood.
▰ Serial dilutions of the specimen - added on to the molten agar.
▰ After being cooled and solidified - Petri dishes are incubated - colony count
is estimated.
91
Stroke Culture
▰ Carried out on agar slopes or slants by

streaking the straight wire in a zigzag fashion.
▰ Used for biochemical test such as urease test.
92
Stab Culture
▰ Made by stabbing the semisolid agar butt by a

straight wire. It is
▰ Used for motility testing using mannitol

motility medium.
93
Incubatory Conditions
▰ Most pathogenic bacteria - aerobes or facultative anaerobes - grow best at

37°C.
▰ Inoculated culture plates - incubated at 37°C aerobically for overnight in

an incubator.
94
Bacteriological Incubator
▰ Device used to incubate culture plates,

biochemical tests and AST plates.
▰ Incubator maintains optimal temperature.
▰ Some incubators are specially designed to

maintain other conditions - humidity and
CO2.
95
Other Incubatory Conditions
For capnophilic bacteria: Candle jar is used
▰ Inoculated media - placed inside a jar with a
lighted candle - burning candle reduces oxygen to
a point where the flame goes off.
▰ Provides an atmosphere of approximately 3–5%
CO2 .
▰ Useful for capnophilic bacteria - Brucella,
Streptococcus, pneumococcus and gonococcus.
96
Other Incubatory Conditions (Cont..)
For microaerophilic bacteria - Campylobacter and Helicobacter - require 5%

oxygen for optimum growth
For obligate anaerobes - anaerobic culture methods are used
97
Anaerobic Culture Methods
▰ Obligate anaerobic bacteria - grow only in the absence of oxygen.
▰ Anaerobic culture methods includes:
 Evacuation and Replacement
 Absorption of Oxygen by Chemical Methods
 Anaerobic Glove box and Anaerobic Work Station
 Reducing Agents
 Pre-reduced Anaerobically Sterilized (PRAS)
98
Evacuation and Replacement
▰ Involves evacuation of the air from jar and replacement with inert gas like
hydrogen followed by removal of the residual oxygen by use of a catalyst.
It is
▰ Carried out either by:
 Manual method – using McIntosh and Filde’s anaerobic
 Automated system (Anoxomat)

99
Evacuation and Replacement (Cont..)
▰ Manual method- McIntosh and

Filde’s anaerobic jar - most popular
method for creating anaerobiosis in
the past, now not in use.
100
Evacuation and Replacement (Cont..)
▰ Automated system (Anoxomat):

Automatically evacuates air - replaces by
hydrogen gas from a cylinder.
▰ Catalyst - sachet containing aluminum

pellets coated with palladium.
101
Absorption of Oxygen by Chemical Methods
GasPak system (BD diagnostics):
▰ Principle - oxygen is removed by chemical reactions.
▰ Uses - sachet containing sodium bicarbonate and sodium borohydride -

react chemically in presence of water - produce hydrogen and CO2 gas
▰ Traces of oxygen - removed by same catalyst used for Anoxomat.
102
Absorption of Oxygen by Chemical Methods (Cont..)
GasPak system (BD diagnostics) (Cont..):
▰ Traces of oxygen - removed by same

catalyst used for Anoxomat.
103
GasPak system (BD diagnostics) (Cont..):

▰ Indicator of anaerobiosis:
 Chemical indicator: Reduced methylene blue - remains colorless in

anaerobic conditions - turns blue on exposure to oxygen.
 Biological indicator – Pseudomonas - absence of growth indicates

perfect anaerobiosis.
104
GENbag (bioMérieux):
▰ Consists of an airtight transparent bag with a generator sachet - rapidly

produces carbon dioxide and creates anaerobic environment.
105
Anaerobic Glove box and Anaerobic Work Station
▰ Provide facility for easy processing,

incubation and examination of the
specimens without exposure to
oxygen
106
Reducing Agents
▰ Oxygen in culture media - reduced by various reducing agents - glucose,

thioglycollate, cooked meat pieces, cysteine and ascorbic acid.
▰ Robertson cooked meat broth - most widely employed anaerobic culture
▰ medium - uses chopped meat particles (beef heart) as reducing agent.
107
Pre-reduced Anaerobically Sterilized (PRAS)
▰ Prepared entirely under oxygen-free conditions from initial sterilization to

packaging in sealed foil packets.
108
Colony Morphology
▰ After overnight incubation - culture media are removed from the incubator
- examined under bright illumination.
▰ Appearance of bacterial colony on culture medium - characteristic for

many organisms - helps in their preliminary identification.
109
Colony Morphology (Cont..)
▰ Size - in millimetres e.g. pin head size is characteristic of staphylococcal
colony & pin point size is characteristic of streptococcal colony.
▰ Shape - circular or irregular
▰ Consistency - dry, moist or mucoid
▰ Density - Opaque, translucent or transparent
▰ Hemolysis on blood agar

110
Color of the colony:
▰ Colored due to - certain properties of the media or organisms.
▰ Eg - Pink colonies produced by lactose fermenters on MacConkey agar and

black colonies by Corynebacterium diphtheriae on potassium tellurite agar.
▰ May also be due to pigment production by the bacteria.
111
Pigment production: Bacteria may produce two types of pigments.
▰ Diffusible pigments
▰ Non-diffusible pigments
112
Hemolysis on Blood Agar
▰ Partial or α hemolysis
▰ Complete or β hemolysis
▰ No hemolysis (γ hemolysis, a misnomer)
113
Culture Smear and Motility Testing
▰ Colonies grown on the culture media - subjected to Gram staining and

motility testing by hanging drop method.
114
CULTURE
IDENTIFICATION
115
CULTURE IDENTIFICATION
Identification of bacteria from culture is made either by:
▰ Conventional biochemical tests or
▰ Automated identification systems.
116
Biochemical Identification
Based on the type of colony morphology, Gram- staining appearance -

appropriate biochemical tests are employed.
▰ Initially - catalase and oxidase tests are done.
117
Biochemical Identification (Cont..)
▰ For gram-negative bacilli - Common biochemical tests done routinely –
‘ICUT’
 Indole test
 Citrate utilization test
 Urea hydrolysis test
 Triple sugar iron test (TSI).
118
▰ For gram-positive cocci:
 Coagulase test (for Staphylococcus aureus)
 CAMP (Christie-Atkins-Munch-Petersen) test - group B Streptococcus.
 Bile esculin hydrolysis test (for Enterococcus)
 Inulin fermentation (for pneumococcus)
 Bile solubility test (for pneumococcus)
119
▰ Antimicrobial susceptibility tests - for bacterial identification:
 Optochin susceptibility test - pneumococcus (sensitive)
 Bacitracin susceptibility test - differentiate group A (sensitive) from

group B (resistant) Streptococcus.
120
Catalase Test
▰ When a drop of hydrogen peroxide (3% H2O2 ) -

added to a colony of any catalase producing
bacteria - effervescence or bubbles appear due to
breakdown of H2O2 by catalase to produce
oxygen.
▰ Differentiate - Staphylococcus (positive) from

Streptococcus (negative). Essentials of Medical Microbiology 121
Oxidase Tests
▰ Detects the presence of cytochrome oxidase enzyme in bacteria which
catalyses the oxidation of reduced cytochrome by atmospheric oxygen.
▰ Oxidase positive (deep purple)- Pseudomonas , Vibrio, Neisseria, Bacillus

etc.
▰ Oxidase negative (no colour change) - members of family
Enterobacteriaceae, Acinetobacter, etc.

122
Indole Test
▰ Detects the ability of certain bacteria to produce enzyme tryptophanase -

breaks down amino acid tryptophan present in the medium into indole.
▰ Indole positive - Red colored ring is formed near the surface of the broth.
Eg: Escherichia coli, Proteus vulgaris, Vibrio cholerae, etc.
123
Indole Test
▰ Indole negative - Yellow colored ring is

formed near the surface of the broth, e.g.
Klebsiella pneumoniae, Proteus mirabilis,
Pseudomonas, Salmonella, etc.
124
Citrate Utilization Test
▰ Detects the ability of a few bacteria to utilize citrate as the sole source of
carbon for their growth, with production of alkaline metabolic products.
▰ Performed on Simmon’s citrate medium - Citrate utilizing bacteria

produce growth and a color change, i.e. original green color changes to blue
125
Citrate Utilization Test (Cont..)
▰ Citrate test - positive for Klebsiella

pneumoniae, Citrobacter, Enterobacter,
etc.
▰ Negative for Escherichia coli, Shigella,

etc.
126
Urea Hydrolysis Test
▰ Urease producing bacteria - split urea present in the medium to produce

ammonia - makes the medium alkaline.
▰ Test - done on Christensen’s urea medium - contains phenol red indicator

- changes to pink color in alkaline medium
127
Urea Hydrolysis Test (Cont..)
▰ Urease test is positive for - Klebsiella

pneumoniae, Proteus species, Helicobacter
pylori, Brucella, etc.
▰ Urease test is negative for - Escherichia coli,

Shigella, Salmonella, etc.
128
Triple Sugar Iron (TSI) Agar Test
▰ Contains three sugars - glucose, sucrose and lactose in the ratio of 1:10:10
parts.
▰ Uninoculated TSI medium - red in color
▰ After inoculation - the medium is incubated at 37°C for 18–24 hours.
129
Triple Sugar Iron (TSI) Agar Test (Cont..)
Interpretation: detects three properties of bacteria, such as
▰ Fermentation of sugars to produce acid
▰ Fermentation of sugars to produce gas
▰ Production of H2 S.
130
131
Reactions in TSI Examples
Acidic slant/acidic butt ≥2 sugars fermented - (1) glucose, (2) lactose or/and
sucrose
A/A, gas produced, no H2S (Fig B) Escherichia coli, Klebsiella pneumoniae
Alkaline slant/acidic butt Only glucose-fermenter group
K/A, no gas, no H2S (Fig C) Shigella
K/A, no gas, H2S produced (small amount) Salmonella Typhi
(Fig D)
K/A, no gas, H2S produced (abundant) (Fig E) Proteus vulgaris
K/A, gas produced, H2S produced Salmonella Paratyphi B
(abundant)
K/A, gas produced, no H2S Salmonella Paratyphi A

Alkaline slant/alkaline butt Non-fermenters group
K/K, no gas, no H2S (Fig F) Pseudomonas, Acinetobacter 132
Automated Systems for Bacterial
Identification
Advantages
▰ Produce faster result
▰ Identify wide range of organisms with accuracy, which are otherwise

difficult to identify (e.g. anaerobes) through conventional biochemical
tests.
133
Automated Systems for Bacterial
Identification (Cont..)
▰ MALDI–TOF (Matrix-assisted laser desorption/ionization time-of-flight)
▰ VITEK 2 (bioMérieux) - automated identification and antimicrobial
susceptibility test.
▰ Phoenix (BD Diagnostics) - automated identification and antimicrobial
susceptibility test
▰ MicroScanWalkAway system (Beckman Coulter) - automated
identification and antimicrobial susceptibility test.
134
MALDI-TOF
▰ Can identify bacteria, fungi, and mycobacteria with a turnaround time of

few minutes and with absolute accuracy.
▰ Two systems are commercially available: VITEK MS (bioMérieux) and

Biotyper system (Bruker).
135
MALDI-TOF (Cont..)
Principle
▰ Examines the pattern of ribosomal proteins present in the organism.
▰ Sample preparation: The colony of an organism - smeared onto a well of

the slide and one drop of matrix solution (composed of cyano-hydroxy-
cinnamic acid) - added to the same well and mixed - slide is loaded in the
system.
136
MALDI-TOF (Cont..)
▰ Steps after loading: three steps occurring in three chambers of the system.
 1. Ionization chamber
 2. Analyzer
 3. Detector
137
MALDI-TOF (Cont..)
138
VITEK 2 automated system
▰ Perform both identification and antimicrobial susceptibility testing (AST)

of bacteria and yeast.
▰ Principle of VITEK for identification: Uses colorimetric reagent card

containing 64 wells; each well contains an individual test substrate.
▰ Result of identification - usually available within 4–6 hours.
139
VITEK 2 automated system (Cont..)
140
ANTIMICROBIAL
SUSCEPTIBILITY TEST
141
ANTIMICROBIAL SUSCEPTIBILITY
TEST
▰ Bacteria exhibit great strain variations in susceptibility to antimicrobial
agents - AST plays a vital role to guide the clinician for tailoring the
empirical antibiotic therapy to pathogen-directed therapy.
▰ Performed only for pathogenic bacteria isolated from the specimen, and
not for the commensal bacteria.
▰ Classified into phenotypic and genotypic methods.

142
ANTIMICROBIAL SUSCEPTIBILITY
TEST (Cont..)
Phenotypic methods -
▰ Disk Diffusion Method- e.g. Kirby–Bauer’s disk diffusion (DD) test
▰ Dilution tests: Broth Dilution and agar dilution Method
▰ Epsilometer or E-test
▰ Automated AST-e.g. Vitek, Phoenix and Microscan systems
Genotypic methods -
▰ PCR detecting drug resistant genes
143
Procedure (Colony disk diffusion)
▰ Antibiotic disks - impregnated on to a suitable medium lawn cultured with
the test isolate.
Antibiotic disks:
▰ Antibiotic disks - commercially or prepared in-house.
▰ Sterile filter paper disks - 6 mm diameter are impregnated with standard

quantity of antibiotic solution.
144
Procedure (Colony disk diffusion) (Cont..)
Medium:
▰ Mueller–Hinton agar (MHA) - standard medium used for AST.
▰ S. pyogenes and S. pneumoniae - Mueller–Hinton blood agar (MHBA)

containing 5% of sheep blood is used.
145
Inoculum preparation:
▰ Directly suspending the colony in the normal saline or
▰ Inoculating into suitable broth and incubating at 37°C for 2 hours
Turbidity:
▰ Adjusted to 0.5 McFarland opacity standard - equivalent to approximately

1.5 × 108 cfu/mL of bacteria.
146
Lawn culture:
▰ Broth - inoculated on to the medium by spreading with sterile swabs
Disks impregnation:
▰ Disks - placed atleast 24 mm (center to center) apart on the MHA plate.
▰ Maximum up to 6 disks can be applied on a 100 mm plate.
147
Interpretation
▰ Susceptibility to the drug - determined by the zone of inhibition of

bacterial growth around the disk - measured by Vernier caliper.
148
Commonly used disk concentrations and interpretation of disk
diffusion test (as per CLSI 2020 guideline)
Antimicrobial agents Disk strength Zone diameter break points (mm) MIC breakpoints (µg/ml)
(µg)
Sensitive Intermediate Resistant Sensitive Intermediate Resistant
Ceftazidime 30 ≥ 21 18-20 ≤ 17 ≤4 8 ≥ 16
Ceftriaxone 30 ≥ 23 20-22 ≤ 19 ≤1 2 ≥4
Ciprofloxacin 5 ≥ 26 22-25 ≤ 21 ≤ 0.25 0.5 ≥1
Cefoxitin (S.aureus) 30 ≥ 22 - ≤ 21 ≤4 - ≥8
Levofloxacin (S.aureus) 5 ≥ 19 16 18 ≤ 15 ≤1 2 ≥4
Cotrimoxazole 1.25/23.75 ≥ 16 11-15 ≤ 10 ≤2 - ≥4

(S.aureus)
149
Direct Disk Diffusion Test
▰ Performed when results are required urgently and single pathogenic
bacterium is suspected in the specimen (for positively flagged blood
culture bottle, sterile fluid or urine).
▰ Specimen - directly inoculated - on to the surface of an agar plate and the

antibiotic disks are applied.
150
Direct Disk Diffusion Test (Cont..)
▰ Results of the direct-DD test - verified by performing AST from the isolate
separately.
151
Dilution Tests
▰ Antimicrobial agent - serially diluted - each dilution is tested with the test
organism for ASTand the MIC is calculated.
▰ MIC (minimum inhibitory concentration) - lowest concentration of an

antimicrobial agent that will inhibit the visible growth of microorganism
after overnight incubation.
152
Dilution Tests (Cont..)
▰ Depending upon whether the dilutions of the antimicrobial agent are made
in agar or broth, there are two types of dilution tests.
 Broth Dilution Method
 Agar Dilution Method
153
Broth Dilution Method
▰ Two types:
 Macro broth dilution (performed in tubes)
 Micro broth dilution (performed in microtiter plate).
154
Broth Dilution Method (Cont..)
▰ Serial dilutions of the antimicrobial agent in Mueller Hinton broth are
taken in tubes - each tube is inoculated with a fixed amount of suspension
of the test organism.
▰ MIC - determined by - lowest concentration of the drug at which there is

no visible growth, i.e. broth appears clear
155
156
▰ Minimum bactericidal concentration (MBC) - obtained by subculturing
from each tube (showing no growth) onto a nutrient agar plate without any
antimicrobial agent.
▰ Tube containing the lowest concentration of the drug that fails to show
growth, on subculture, is the MBC of the drug for that test strain.
157
Agar Dilution Method
▰ Serial dilutions of the drug - prepared in molten agar and poured into petri
dishes.
▰ Test strain is spot inoculated.
Advantage:
▰ Several strains can be tested at the same time by using the same plate
▰ Directly measures the MBC

158
Epsilometer or E-test
▰ Uses an absorbent strip containing predefined
gradient (serial dilution) of antibiotic
concentration immobilized along its length.
▰ Applied to a lawn inoculum of a bacterium.
▰ Antibiotic concentration at which the ellipse

edge intersects the strip -MIC value.
159
Automated Antimicrobial Susceptibility Tests
▰ VITEK 2 identification and antimicrobial sensitivity system
▰ Phoenix System (Becton Dickinson)
▰ Micro Scan Walk Away system.
160
VITEK 2 Automated System for AST
▰ Principle - microbroth dilution.
▰ Uses reagent card containing 64 wells - contain doubling dilution of

antimicrobial agents. The
▰ Organism suspension - added to the wells (Figure 3.3.23 and Table 3.3.7)
▰ Cards are incubated in the system at 35.5 ±1°C.
161
VITEK 2 Automated System for AST
(Cont..)
▰ Reading - taken once in every 15

minutes.
▰ Results - available within 8-10 hours for

gram negative bacilli and 16-18 hours
for gram-positive cocci.
162
Role of MIC
▰ To select the most appropriate antibiotic - Lower is the MIC, better is
the therapeutic efficacy
▰ Better guided - calculating the therapeutic index - ratio of susceptibility

breakpoint divided by the MIC of the test isolate.
▰ MIC-guided therapy
163
Interpretation of AST
Terminology Definition
Susceptible (S)
Antibiotic is clinically effective when used in standard therapeutic dose.
Intermediate (I) Antibiotic is not clinically effective when used in standard dose; but may be active
when used in increased dose.
Susceptible dose
Antibiotic will be clinically active only if given in increased dose.
dependent (SDD)
Resistant (R) Antibiotic is NOT clinically effective when used in either standard dose or
increased dose.
164
Choice of Antibiotics to be Included in Panel
▰ Clinically indicated
▰ Organism isolated and local resistance pattern
▰ Intrinsic resistance - excluded from the panel
▰ Antimicrobial agent - oral and parenteral - included
▰ Locally available antibiotic
▰ Site of action - tested which are active in the site.
▰ Predicting susceptibility
165
Selective or Cascade Reporting
▰ Clinical microbiology laboratory - test for the full antibiotic panel, but -
should report only in a selective manner.
▰ If all antimicrobial agents are susceptible - only first-line agents are

reported - second-line and restricted antimicrobials are supressed.
166
Selective or Cascade Reporting (Cont..)
Enterobacteriaceae Antimicrobial agent used in
VITEK
First line Ampicillin
First line Amoxicillin- clavulanic acid
First line Ciprofloxacin
Second line Cefoperazone-sulbactam
Second line Piperacillin- tazobactam
Restricted Meropenem
Restricted Colistin
167
SEROLOGY
168
SEROLOGY
Detection of either antigen or antibody in the serum of the patient, by various

immunological assays such as—
▰ Precipitation
▰ Agglutination
▰ ELISA
▰ Rapid test.
169
MOLECULAR
METHODS
170
MOLECULAR METHODS
▰ Polymerase chain reaction (PCR)

▰ Real-time polymerase chain reaction (rt-PCR)
▰ Loop mediated isothermal amplification (LAMP)
▰ Automated PCR such as Biofire FilmArray
▰ Automated real-time PCR such as cartridge based nucleic acid
amplification test (CBNAAT)
171
Polymerase Chain Reaction (PCR)
▰ Technology in molecular biology used to amplify a single or few copies of

a piece of DNA to generate millions of copies of DNA.
▰ Developed by Kary B Mullis
172
Polymerase Chain Reaction (PCR) (Cont..)
Principle : three basic steps
1. DNA extraction from the organism
2. Amplification of extracted DNA
3. Gel electrophoresis of amplified product:
173
Polymerase Chain Reaction (PCR) (Cont..)
174
Modifications of PCR
1. Reverse transcriptase PCR (RT-PCR)
2. Nested PCR
3. Multiplex PCR
175
Biofire FilmArray
▰ Multiplex nested PCR system
▰ Four panels are available such as

respiratory, gastrointestinal,
meningitis-encephalitis and blood
culture identification panels
176
Real-time PCR (rt-PCR)
▰ PCR technology, which is used to

amplify and simultaneously detect
or quantify a targeted DNA
molecule on real-time basis.
177
Loop Mediated Isothermal Amplification (LAMP)
▰ Isothermal nucleic acid amplification technique.
▰ Amplification is carried out at a constant temperature of 60–65° (in

contrast to alternating temperature cycles in PCR)
178
Nucleic Acid Probes
▰ Radiolabeled or fluorescent labelled pieces of single stranded DNA or

RNA - used for the detection of homologous nucleic acid by hybridization.
▰ Two types of nucleic acid probes—DNA probes and RNA probes.
179
MICROBIAL TYPING
180
MICROBIAL TYPING
Refers to characterization of an organism beyond its species level.
Applications:
▰ Used to determine the relatedness between different microbial strains of the
same species.
▰ Investigate outbreaks
▰ Determine the source and routes of infections
▰ Trace cross-infection
▰ Differentiate virulent strains from avirulent strains of same species
181
MICROBIAL TYPING (Cont..)
Classification:
▰ Phenotypic methods
▰ Genotypic methods.
182
Characteristics of Typing Methods
▰ Typeability
▰ Reproducibility
▰ Discriminative power
▰ Practicality
183
Phenotypic Methods
Bacteriophage typing: Strains of an organism - further differentiated into

subspecies level based on their susceptibility to bacteriophages.
Bacteriocin typing: Bacteriocin - antibiotic like proteinaceous substance -

based on the ability of a strain to produce particular bacteriocin which inhibits
the growth of a set of selected indicator strains.
184
Phenotypic Methods (Cont..)
Biotyping: Refers to intra species classification based on different

biochemical properties of the organism.
Antibiogram typing: Classifies the organism into different groups based on

their resistance pattern to different antimicrobials.
Serotyping: Typing method based on the antigenic property of an organism
185
Genotypic Methods
▰ Restricted Fragment Length Polymorphism (RFLP)
▰ Ribotyping
▰ Pulse Field Gel Electrophoresis (PFGE)
▰ Amplified Fragment Length Polymorphism (AFLP)
▰ Sequencing-based Methods
186
Questions:
▰ Q1. Agar concentration required to prepare semisolid media:
a. 0.1%
b. 0.5%
c. 2%
d. 6%
187
Questions:
▰ Q2. Type of specimen collected for URTI:
a. Blood
b. Throat swab
c. Sputum
d. BAL
188
Questions:
▰ Q3. Sulfuric acid (%) needed for decolorization of M. tuberculosis:
a. 25%
b. 5%
c. 1%
d. 0.5%
189
Questions:
▰ Q4. Examples of enriched media are all except:
a. Blood agar
b. Loeffler’s serum slope
c. Blood culture media
d. MacConkey agar
190
Questions:
▰ Q5. Automated blood culture bottles contains:
a. Selenite F broth
b. Tryptic soy broth
c. Peptone water broth
d. BHI broth
191
Questions:
▰ Q6. Which of the following is not an equipment for automated blood

culture:
a. BacT/ALERT
b. VITEK2
c. BACTEC
d. VersaTREK
192
Questions:
▰ Q7. BacT/ALERT - principle is based on::
a. Calorimetric detection
b. Fluorescent detection
c. Detection of change of pressure
d. All the above
193
Questions:
▰ Q8. Three basic steps of PCR cycle involves all, except:
a. Denaturation
b. Amplification
c. Extension
d. Gel documentation
194

Chapter 3.3-Lab Diagnosis of Bacterial Infection

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GENERAL BACTERIOLOGY:

At the end of the session, the students will be able to understand:

▰ Direct Detection, Staining Techniques

▰ Culture, Identification and AST

Blood stream infection, sepsis, endocarditis Paired blood culture specimens

Meningitis Cerebrospinal fluid (CSF)

Urinary tract infections Midstream urine

Following principles should be followed while collecting the specimen:

▰ Before antibiotics start

▰ Contamination with indigenous flora should be avoided

▰ Swabs - convenient but considered inferior to tissue, aspirate and body

▰ Container: sterile, tightly sealed, leak proof, wide-mouth

▰ Labeling: All specimens - labelled with name, age, gender, treating

▰ Rejection: Specimens contaminated or improperly labelled may be rejected

▰ Anaerobic culture - proper anaerobic collection containers with media

▰ Immediate transport (<15 minutes) - CSF and body fluids, ocular

▰ Urine (midstream) - added with preservative (boric acid) - transported

▰ Stool culture - transported within 1 hour.

▰ Rectal swabs - up to 24 hours is acceptable.

▰ For anaerobic culture - Robertson’s cooked meat broth or any specialized

Most specimens - stored at room temperature - up to 24 hours.

There are some exceptions:

▰ Blood cultures - incubated at 37°C immediately up on receipt.

▰ Sterile body fluids - immediately plated up on receipt - incubated at 37°C.

▰ Corneal scrapping - plated at bed-side.

▰ Stool culture - stored up to 72 hours at 4°C

▰ Urine (mid-stream and from catheter), lower respiratory tract

▰ It is necessary to use staining methods to produce color contrast and

▰ Fixation - protects the internal structures of cells.

▰ Done by two methods.

1. Heat fixation: Gentle flame heating an air-dried film of bacteria.

2. Methanol fixation—used for blood smears.

▰ Negative staining: A drop of bacterial suspension is mixed with dyes

▰ Impregnation methods: Bacterial cells and structures are thickened by

Christian Gram (1884).

▰ Gram stain still remains the

primary stain i.e. violet.

▰ Gram-negative bacteria are decolorized

▰ Gram-positive cell wall - thick peptidoglycan layer (50–100 layers thick).

▰ Acts as permeability barrier preventing loss of crystal violet.

▰ Alcohol is thought to shrink the pores of the thick peptidoglycan.

▰ Thin peptidoglycan layer in gram-negative cell wall - is not tightly cross

▰ Presence of lipopolysaccharide layer in the cell wall of gram-negative

▰ Identification of Gram staining from bacterial culture helps in further

▰ Initiation of empirical treatment with broad spectrum antibiotics - started

▰ Early presumptive identification of fastidious organisms - Haemophilus.

▰ Helps in screening the quality of the sputum specimen before processing it

▰ Discovered by Paul Ehrlich and subsequently modified by Ziehl and

▰ Acid fastness - due to presence of mycolic acid in the cell wall.

▰ Smear = 2 × 3 cm in size from the yellow purulent portion of the sputum.

▰ Step 1 (primary stain) - Carbol fuchsin (1%) for 5 minutes. Intermittent

▰ Step 2 (decolorization) - 25% sulfuric acid for 2–4 minutes.

▰ Step 3 (counter staining) - 0.1% methylene blue slide for 30 seconds.

▰ Cold method (Kinyoun’s method): Intermittent heating is not required.

▰ Acid-alcohol can be used as decolorizer alternatively.

▰ Malachite green can be used as counter stain.

▰ Concentration of sulfuric acid - vary depending on the acid-fastness of the

▰ Used to demonstrate - metachromatic granules of Corynebacterium

Composition of Albert stain includes: