1.
LABORATORY DIAGNOSIS
ANILA P J
MSC MEDICAL MICROBIOLOGY
JSS MEDICAL COLLEGE

2.
APPROACH TO
LABORATORY
DIAGNOSIS
• The laboratory diagnosis of infectious diseases
involves two main approaches:
• Bacteriological approach:- Identified by
microscopic observation, macroscopic
identification, biochemical reactions and
molecular diagnosis approaches.
• Immunological approach:- Identified by
detection of antibodies against organism
in the patient’s serum.

3.
Lab diagnosis is useful for :
• Identification: causative agent responsible for disease.
• Treatment : accurate antimicrobial therapy.
• Surveillance purpose : to assess the disease burden in the community by
estimating the prevalence and incidence of the infections.
• For outbreak investigation : diphtheria outbreaks in the community, MRSA
outbreak in the hospitals.
• To start PEP(post-exposure prophylaxis): for infectious diseases such as
anthrax, and plaque.

4.
TEST CYCLE
Preanalytical
(sampling, labelling, transport, storage, specimen clinical information)
Analytical
(macroscopic, microscopic, culture, identification, interpretation, antibiogram,…)
Postanalytical
(final report to physician)

5.
• Laboratory diagnosis comprises of different steps :
• Specimen collection
• Direct detection (microscopy )
• Culture
• Identification (biochemical tests, automated
identification)
• Antimicrobial susceptibility test
• Serology
• Molecular methods

6.
A) SPECIMEN COLLECTION
• Depends upon the type of underlying infections.
• Proper collection of sample is of paramount importance for the isolation of the
bacteria in culture.

7.
GENERAL PRINCIPLES
• Standard precautions should be followed for collecting and handling all
specimens.
• Should collect before antibiotic starts
• Contamination with indigenous flora should be avoided.
• Swabs are though convenient but considered inferior to tissue, aspirate and body
fluids.
• Container : sterile, tightly sealed, leakproof, wide mouth, screw capped
containers should be used.

8.
• Labelling : with name, age, gender, treating physician, diagnosis, antibiotic
history, type of specimen and desired investigation name.
• Rejection : if contaminated or compromised or improperly labelled.
• If anaerobic culture is requested, proper anaerobic collection containers with
media should be used.
• Specimen should not be sent in container containing formalin for microbiological
analysis.

9.
TYPES OF INFECTIONS SPECIMEN COLLECTED
Bloodstream infection, sepsis,
endocarditis
Paired blood culture specimen(8-10 ml)
Infectious disease requiring serology Blood (2ml/investigation)
Diarrheal diseases Stool, rectal swab
meningitis CSF
Infections of other sterile body area Eg., pleural fluid, synovial fluid, peritoneal fluid
Skin & soft tissue infections Pus or exudate, wound swabs, aspirates from abscess and tissue
bits.
Anaerobic infections Aspirates, tissues, blood & sterile body fluid, bone marrow.
URT infections Throat swab, nasopharyngeal swab, per-nasal swab.
LRT infections Sputum, endotracheal aspirate, BAL, protected specimen brush
UTI Midstream urine, suprapubic aspirate, urine from catheter tube
Genital infections Urethral swabs, cervical swabs, exudate from genital ulcers.
Eye infections Conjunctival swabs, corneal scrapings, aqueous or vitreous fluid.
Ear infections Swabs from outer ear, aspirate from inner ear.

10.
Specimen transport:-
• Most of specimen, should not exceed 2 hours; exception:-
Immediate transport (<15 min) include CSF, and body fluids, ocular specimens,
tissue specimens, suprapubic aspirate, bone specimen.
Urine added with preservative (boric acid) - up to 24 hours.
Stool culture: transported with in 1 hour, but with transport medium - up to 24hrs.
Rectal swabs – up to 24hrs.
For anaerobic culture: specimen put into RCMB or any other anaerobic transport
medium and transported immediately.

11.
SPECIMEN STORAGE:-
• Can be stored at room temperature immediately after receipt, Up to 24 hrs.
exceptions are :
Blood cultures – incubated at 37°C .
Sterile body fluids, bone, vitreous fluid , suprabic aspirate – immediately
plated or incubated 37°C.
Corneal scraping – immediately plated at bed-side on to BA or CA.
Stool culture – stored up to 72hrs at 4°C.
Urine, LRT specimen, gastric biopsy – stored up to 24 hrs at 4°C.

12.
DIRECT METHOD
• Microscopic detection:-
 bacterial motility :–
* Hanging drop method

13.
 Morphology and staining reaction:-
SIMPLE STAIN: methylene blue stain

14.
• NEGATIVE STAINING :- nigrosine/ India ink
Demonstration of bacterial/yeast capsules which do not take up simple stain.
E.g., S.pneumoniae, Cryptococcus

15.
• GRAM STAIN:- differentiate between G+ve and G-ve bacteria.

16.
• Uses of gram stain:-
o To differentiate G+ve and G-ve
bacteria.
o To start empirical treatment
o For identification of fastidious org.,
which takes time to grow on
culture.
o For anaerobic organisms which do
not grow on ordinary medium.
o Analysing quality of specimen(
sputum specimen)
Gram positive organisms:-
Staphylococcus
Streptococcus
Enterococcus
Bacillus
Clostridium
Candida
Gram negative organisms:-
Neisseria
Shigella
Salmonella
Escherichia coli
Klebsiella
Vibrio

17.
ACID-FAST STAIN:- Diffrentiate acid fast org. from non acid-fast org.
Acid-fast organism

18.
Modifications :
o cold method (kinyoun’s method) –
intermittent heating is not needed.
o acid-alcohol alternatively used as
decolourizer.
o malachite green used as counter stain.
o Different con. Of sulphuric acid.
Acid fast organisms :-
Mycobacterium tuberculosis
Mycobacterium leprae
Nocardia
Bacterial spores

19.
• ALBERT STAIN : demonstrate the metachromatic granules of
Corynebacterium diphtheriae.
Green bacilli with bluish black
metachromatic Granules of C. diphtheriae

20.
CULTURE
• Specimens are inoculated on to various
culture media and incubated.
• Colonies grown are subjected to
identification and AST.
• Different culture media:-
• SIMPLE/BASAL MEDIA:- support
growth of non-fastidious bacteria.
*Peptone water
*Nutrient broth
*Nutrient agar

21.
• ENRICHMENT MEDIA :-liquid media added with
inhibitory agents which selectively allow org. to
grow.
tetrathionate broth – salmonella typhi
selenite-F broth – isolation of Shigella
alkaline peptone water – isolation of Vibrio cholerae

22.
MEDIA ISOLATION
Lowenstein-Jensen
medium
Mycobacterium
tuberculosis
Thiosulphate citrate
bilesalt sucrose agar
Vibrio cholerae
Deoxycholate citrate
agar
Salmonella & shigella
Xylose lysine
deoxycholate agar
Salmonella & Shigella
Potassium tellurite
agar
Corynebacterium
diphtheriae

23.
• TRANSPORT MEDIA :- transport of clinical specimen
suspected to contain delicate organism or when delay is
expected.
Organism Transport medium
Neisseria Amies medium and Stuart's medium
Vibrio cholerae V R medium
Autoclaved sea water
Cary Blair medium
Shigella, salmonella Buffered glycerol saline
Cary Blair medium

24.
• DIFFERENTIAL MEDIA :- differentiate between 2 groups of bacteria
using an indicator.
MacConkey agar – Differential and low selective medium.
Isolation of enteric gram negative bacteria.
Differentiate lactose fermenters (pink colonies)
and Non lactose fermenters (colorless colonies).
Indicator – neutral red
CLED(cysteine lactose electrolyte deficient agar) –
Differentiate between LF and NLF colonies.
For urine specimen.

25.
• ANAEROBIC CULTURE MEDIA:-
contain reducing substances which
take up oxygen & create lower redox
potential and allows growth of
obligate anaerobes.
o Robertson cooked meat broth
o Brain heart infusion broth
o Egg yolk agar
o Thioglycollate broth

26.
BLOOD
CULTURE
• Recovery of bacteria from blood is difficult as
they are in lesser quantity in blood and many
are fastidious; so enriched media are used.
• Two types
1) Conventional blood culture
2) Automated blood culture

27.
Conventional blood culture media
• Monophasic medium – contain
BHI broth
• Biphasic medium – liquid phase
with BHI broth solid agar slope
with BHI agar
• Disadvantage : -
subcultures are made onto BA
and MA periodically for one
week.
Higher risk of contamination
due to opening of cap everytime
when subcultures are made

28.
Automated
blood
culture
technique
Continuous
automated
monitoring.
Incubated bottles are
periodically tilted
automatically.
Once positive for
microbial growth,
instrument gives
signal.
Composition – tryptic
soy broth / BHI broth,
polymeric resin
beads
Specimens – blood,
CSF, synovial fluid,
pleural fluid
Gives higher yield of
positive culture
It is Rapid
Less labor intensive –
fully automated.

29.
Automated system
1) BacT/ALERT 3D
2) BacT/ALERT VIRTUO
3) BACTEC
Disadvantages:-
• High cost of instrument and culture
bottles.
• Inability to observe the colony
morphology.

30.
Culture method
• It involves inoculating specimen onto
appropriate culture media, incubating culture
plates in appropriate conditions.
1.Selection of media:-
depends upon type of specimen to be
processed.
commonly used are BA & MA.
2. Inoculation of specimen:-
with the help of bacteriological loop (platinum
or nichrome wire)
loop is heated till red hot and made cool.

31.
Inoculation method
1.STREAK CULTURE
• Used for obtaining individual
isolated colonies from a mixed
culture.
• Intermittent heating is done
between the streak.

32.
2. LIQUID CULTURE
Bacterial growth is detected by observing
turbidity in the medium.
Advantages:
• Specimen containing small quantity of bacteria
• Specimen containing antibiotics
• When large yield of bacteria are required
Disadvantages:
• Do not provide pure culture
• No visible colonies

33.
LAWN CULTURE
For antimicrobial susceptibility testing by disc diffusion
method by using swab
POUR PLATE TECHNIQUE
For quantifying bacterial load
Serial dilutions are added onto molten agar
STROKE CULTURE
Carried out on agar slope by streaking in zig zag fashion
STAB CULTURE
Stabbing semi solid agar butt

34.
ANAEROBIC CULTURE METHOD
• McIntosh and Filde’s anaerobic jar
• Anoxomat
• GasPak system
• GENbag
• Anaerobic glove box and anaerobic
workstation
• Reducing agents – RCM broth.
• Pre-reduced Anaerobically Sterilized
media

35.
Incubatory condition
• Aerobes and facultative anaerobes – 37°C aerobically
overnight in an incubator.
• For capnophilic bacteria – candle jar is used.(3-5%
CO2)
• For microaerophilic bacteria – requires 5% O2.
• For obligate anaerobes – anaerobic culture methods
used.

36.
COLONY MORPHOLOGY
After overnight incubation examined under
bright illumination for :
• Size (mm)
• Shape - circular/irregular
• consistency - dry , moist or mucoid
• Density - opaque, translucent or transparent
• Haemolysis on BA
• Colour of the colony
• Pigment production

37.
Culture smear &
motility testing
Colonies grown on culture media are
subjected to gram staining and
motility testing using hanging drop
method.

38.
Culture
Identification
Two types:
Biochemical
identification
Automated
identification system
Identification of bacteria from culture
is made either by conventional
biochemical tests or by automated
identification system.

39.
Biochemical identification
2. OXIDASE TEST
Detect presence of cytochrome oxidise enzyme.
It turns deep purple with in 10 sec.
* Positive - Vibrio, Pseudomonas etc.
* Negative - Enterobacteriaceae family etc.
1. CATALASE TEST
Catalase producing bacteria mixed with a drop of 3%
hydrogen peroxide & look for effervescence
* Positive – Staphylococcus, Enterobacteriaceae family etc.
* Negative – Streptococcus

40.
4. CITRATE UTILISATION TEST
• Ability of bacteria to utilise citrate as sole source of
carbon
• Medium – simmon’s citrate medium
• Indicator – bromothymol blue
• Produce colour change from green to blue
• Positive – klebsiella, Citrobacter
• Negative – E.coli, shigella

41.
5. UREA HYDROLYSIS TEST
• Ability of bacteria to split urea to
produce ammonia and makes medium
alkaline
• Medium – Christensen urea medium
• Indicator – phenol red
• Colour changes to pink.
• Positive – Klebsiella, Proteus
• Negative – E.coli , Shigella

42.
6. TRIPLE SUGAR IRON AGAR TEST
Contains 3 sugars – glucose, sucrose, lactose (1:10:10)
Ability of organism to ferment sugars and to produce
hydrogen sulphide.
Medium – TSI medium
Indicator – phenol red
Interpretation :-
A/A – (acid/acid) both slant & butt change to yellow.
K/A – alkaline slant (red)/acid butt (yellow)
K/NC – Alkaline slant (red) /Alkaline butt (red)
Gas production – cracks on medium
H2S production – medium changes to Black.

43.
AUTOMATED
SYSTEM
1. MALDI-TOF
2. VITEK 2
3. Phoenix
4. Microscanwalkaway system

44.
ANTIMICROBIAL
SUSCEPTIBILITY
TEST
• Done for pathogenic bacteria.
• Types:
1. Disc diffusion method (kirby-Bauer’s method)
2. Dilution test (broth & agar dilution)
3. E-test
4. Automated AST (VITEK)

45.
DISK DIFFUSION
METHOD
• Most widely used method
• Suitable for rapidly growing pathogenic bacteria
• Mostly performed from colony
• Medium – Mueller-Hinton agar(MHA)
• Inoculum – suspending colony in normal saline or inoculating into suitable
broth & incubating at 37°C for 2 hrs

46.
• Turbidity – matching with 0.5 McFarland
standard(1.5 X 108CFU/ml)
• Lawn culture done using swabs.
• Antibiotic discs placed at least 24 mm apart(6 discs in
100 mm plate )
• Incubation – 37°C for 16 to 18 hrs.

47.
• Interpretation :-
• Antibiotics in the disk diffuses through the
solid medium; conc. is highest near the site
of application of antibiotic disk and
decreases gradually away from it.
• Susceptibility is determined by zone of
inhibition of bacterial growth around the disk
& is measured using vernier caliper.
• Interpretation of zone into sensitive,
intermediate or resistant is based on the
standard zone size interpretation chart,
provided by CLSI or EUCAST guidelines

48.
• Automated Antimicrobial susceptibility testing:-
 work by the principle of micro broth dilution.
 Use commercially available panels.
 Provide more rapid results.
o VITEK 2
o Phoenix system
o micro scan walk away system

49.
SEROLOGY
• Include detection of either antigen or
antibody in the serum of patient by
various immunological assays –
precipitation, agglutination, ELISA &
rapid test.
• Various serological tests are:
o widal test – enteric fever
o Standard agglutination test –
Brucellosis
o Weil-felix test – Rickettsial
infection
o VDRL or RPR - Syphilis

50.
MOLECULAR METHODS
Amplification of nucleic acid based:
• Polymerase chain reaction(PCR)
• Real-time PCR (rt-PCR)
• Biofire Film Assay
• CBNAAT
Non-amplification methods :
• DNA hybridisation method
• (line probe assay)

51.
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