This document discusses various techniques used to identify and diagnose microorganisms and diseases. It begins by describing microscopic organisms and methods to identify microbes, including staining methods, biochemical tests, PCR, and spectrometric techniques. It then provides details on specific diagnostic techniques for diseases like typhoid, tuberculosis, malaria, cholera, hepatitis, meningitis, syphilis, gonorrhea, HIV/AIDS and diphtheria. These include culture-based techniques, molecular tests like PCR, serological tests, and rapid diagnostic tests. The document emphasizes the importance of accurate identification of pathogens for effective disease diagnosis and treatment.

1. Identification, Cultivation,
&
Handling of microorganisms
Recent microbial techniques
developed for diagnosing
some common
diseases.
&
Part-1
Part-2
Presented by
Dr. Karunanidhan
Pharm D 2nd yr

2. Identification, Cultivation,
&
Handling of microorganisms
Part-1
Presented by
Dr. Karunanidhan
Pharm D 2nd yr

3. MICROORGANISMS
A microorganism, or microbe-
Is a microscopic organism,
May exist in its single-celled form or in a colony of cells.
A microorganism or microbe is an organism that is so small that it is
microscopic (invisible to the naked eye).

4. IDENTIFICATION OF MICROORGANISMS
• Identification of microbes is one of the major responsibilities of a microbiologist.
• Various methods are involved in the identification of microorganisms, listed below:
Staining methods (staining reactions).
Biochemical tests.
PCR.
Spectrometric techniques.
Capillary electrophoresis.

5. STAINING METHODS (STAINING REACTIONS)
• Stain is an organic compound commonly called dye that binds to a cellular
structure & gives it respective colour, containing a benzene ring with
chromophore & auxochrome group & this process is called staining.
• Different staining techniques are used for visualization, differentiation,
identification & separation of microbes in the terms of characteristics & cellular
structures.
• Significance of staining:
Increases visibility of specimen.
Highlights specific morphological features.
Preserves specimens.

6. TYPES OF STAINING METHODS (STAINING
REACTIONS)
• SIMPLE STAINING: in simple staining, the bacterial cell is stained with a single stain.
Eg. Methylene blue, crystal violet, carbol fuchsin safranin etc.
 Significance: to elucidate the morphology & arrangement of bacterial cells.
• Negative staining: in this staining, bacteria is mixed with acidic stain & a smear is
prepared. Eg. Eosin & nigrosin. The acidic stain doed not penetrate the cells because
of the –ve charge on the surface of bacteria. Hence, unstained cells are observed
against the coloured background.
 Significance: useful in demonstration of bacterial capsule.

7. • GRAM STAINING: the cells are stained with a basic stain & treated with an iodine –
potassium iodide mixture to fix the stain. Then the stain is washed with alcohol or
acetone (decolorizer) & counterstained with a polar dye of a different color (eg.
Safranin). Gram
–ve bacteria is easily decolorised as red colour. (Eg. Escherichia coli etc).
Significance: used for differentiation & identification of bacteria.
• Acid – fast staining: the strong decolourizer, acid alcohol is added to such a
stained smear, the non- acid fast cells loose the stain & become colourless.
Significance: this method categorises bacteria into acid – fast & non- acid fast.
• SPORE STAINING: the smear is heated for staining the spores. Once, the spores
accepts the malachite green, it cannot be decolourised by the tap water.
Significance: used for detection for spore – carrying bacteria & types of spores.

9. BIOCHEMICAL
TESTSLiving organisms are differentiated on the basis of various enzyme- catalysed
metabolic reactions. Tests include:
SUGAR FERMENTATION.
LITMUS MILK REACTIONS.
INDOLE PRODUCTION.
METHYL RED (MR)TEST.
VOGES – PROSKAUER (VP) TEST.
CITRATE UTILISATION.
 NITRATE REDUCTION TEST.
HYDROGEN SULPHIDE
PRODUCTION.
POTASSIUM CYANIDE TEST.
CATALASE PRODUCTION.
UREASE TEST.
OXIDASE TEST.

10. • LITMUS MILK REACTIONS: The major substrates capable of
transformation are milk sugar lactose & milk forms an
excellent differential medium in which microorganisms can
metabolise milk substrates depending on their enzymatic
complement such as lactose fermentation, gas production,
litmus reduction, curd formation, proteolysis & alkaline
reaction.
• SUGAR FERMENTATION: The ability of the microorganisms
to ferment various sugars is tested by inoculation of the
test microorganisms in different sugar media containing
indicators. Acid production is shown by the change in
colour of medium to pink / red & the gas, if produced,
gets collected in DURHAM’S TUBE.

11. • METHYL RED (MR) TEST/ VOGES- PROSKAUER (VP) TEST : This is
used to detect the production of acid during fermentation of
glucose. By production of acid, ph of medium falls. A red/ pink
colour signifies positive while yellow signifies a –ve test.
• INDOLE PRODUCTION: Indole production is detected by
inoculating the test microorganisms into peptone water &
incubating it at 37 degree celsius for 48- 96 hours then add
0.5ml of kovac’s reagent & mix gently. A red colour in
alcohol layer indicates a positive reaction of production of
indole.

12. • CITRATE UTILISATION: This test is used to study the ability of an microorganisms
to utilise citrate as the sole source of carbon for the growth.
Indole, MR, VP & citrate tests are done in routine for the classification of gram –
negative enteric bacteria. They are commonly referred as IMVIC TESTS.
• UREASE TEST: This test detects the ability of a microorganisms to produce urease
enzyme. This test microorganisms is inoculated on the entire slope of
Christensen's medium which contains urea & phenol red indicator, incubated at
37 degree Celsius & examined after 48 hours & overnight incubation. The latter
in the presence of water converts urea into ammonia & carbon dioxide. Ammonia
makes the medium alkaline & phenol red indicator changes to purple -red in
colour.

13. • CATALASE PRODUCTION: Add a loopful of 10%
hydrogen peroxide on colonies of the test
microorganisms with platinum loop from
nutrient agar plate & dip it in a drop of 10%
hydrogen peroxide on a clean glass slide. The
production of gas bubbles from the culture
indicates a positive reaction.
• OXIDASE TEST: This test depends on the presence of
oxidases that catalyse oxidation of reduced tetra
methyl- p-phenylene diamene dihydro chloride by
molecular oxygen. Add a drop of freshly prepared 1%
of oxidase reagent on a piece of filter paper. Then rub
a few colonies of test microorganisms on it. If it is
oxidase positive, it will produce a deep purple colour
within 10 seconds. Positive test is shown by the
presence of a deep purple colour.

14. • NITRATE REDUCTION TEST: This test detects the
production of enzyme nitrate reductase which
reduces to nitrate to nitrite. A red colour
developing within a few minutes indicates the
presence of nitrite.
BIOCHEMICAL TESTS:
• TO IDENTIFY GRAM
POSITIVE BACTERIA:
• CATALASE TEST.
• NITRATE REDUCTION
TEST.
• COAGULASE TEST.
• TO IDENTIFY GRAM
NEGATIVE BACTERIA:
• UREASE TEST.
• METHYL RED (MR) TEST.
• OXIDASE TEST.

15. Recent microbial techniques
developed for diagnosing
some common
diseases.
Presenting by :-
Karunanidhan
Pharm D 2nd year
Part-2

16. TYPHOID
Causative agent - Salmonella typhi
Diagnostic Techniques :-
 Culture method (in special
medium)
 Blood culture
 Bone marrow culture (most sensitive
test)
 Stool culture
 Urine culture
 Gram staining
 Widal test (serological
test)• Agglutination test which detects the presence of serum agglutinins (H & O) in
patient’s serum with typhoid and para typhoid fever.
• The test undergoes in isotonic saline solution.
• If agglutination occurs in the testing tube, then typhoid is confirmed.
 WHO recommends culture instead of Widal test.
=> After culturing ,biochemical tests are used for confirmation such as – citrate test,
motility test , urease test.

17.  Antimicrobial
susceptibility test
• For initial screening only
• Should be confirmed by Widal
test
• Based on Detection of IgG &
IgM antibodies
Test kits
• By Kirby-Bauer disk diffusion
method
• Using different antimicrobial
agents
• Like ciprofloxacin,
chloramphenicol , etc.
 Rapid test kits
(recent)
C - control
line
T1- IgG Ab
T2- IgM Ab
S - sample

18. TUBERCULOSIS
Causative agent – Mycobacterium tuberculosis
Diagnostic Techniques :-
 Mantoux test/ Tuberculin
skin test
• Test for immunity to T.B. using intradermal
injection of tuberculin (protein of bacteria) in
lower part of arm
• After 48-72hrs , swelling size is measured
• Sometimes, may produce false results; so
expertise & extra care neededAdvanceme
nt- IGRAs (Interferon Gamma Release
Assays)• Fast technique , result in 24hrs
• Blood test that measure immune response to
Mycobacterium
• Measures latent T.B. infection
• Test relies on the fact that T-lymphocytes will
release INF- gamma when exposed to specific

19.  Sputum smear microscopy
• Thin layer of sample is placed on single or double cavity glass slide, called
smear.
• By using different stains, bacteria is identified.
 Fluorescent
microscopy• Makes sputum test more accurate due to larger and clear
viewing angles.
• Mercury vapour lamp is used.
• Fluorochrome dyes are used to stain the smear
• Turn around time reduced.
• Results are effective
 NAAT
• Also known as Nucleic acid amplification test or DNA probe
test
• Based on testing for presence of bacteria
• (details in gonorrhoea. Slide no. 13)
Fluorescent
microscope : a
diagrammatic
representation

20. MALARIA
Causative agent – Plasmodium parasite
Diagnostic Techniques :-
 Thick and thin blood smear (gold
standard test)
 Rapid diagnostic test
(RDT)
Advanceme
nt-
• Also called Antigen testing
• Immuno-chromatographic antigen-detection tests
• Rely on the capture of dye-labelled antibodies to produce a visible band on a strip
of nitro-cellulose.
• Blood drop is put on strip that changes colour to show the presence of parasite.
• Various types of RDTs available for different species. Drug resistance test
 Blood test
 For blood constituent count
 To know how serious the infection is.

21. Thermal cycler
 Molecular test or PCR
• Helps also to identify the
type of parasite.
• Best choice ,if low number
of parasite in blood or if
blood smears are
producing no results
• Effective when , if there is
any change in morphology
of parasites &
microscopist is not able to
identify it.
• Amplified DNA are seen in
U.V. light.

22.  QBC (Quantitative Buffy Coat Test)
• Rapid test for diagnosis of malaria
• Based on acridine orange staining of
centrifuged peripheral blood samples in
a microhematocrit tube.
• Fluorescence microscopy based
diagnostic test
• It speeds & amplifies malaria detection
with a combination of features & benefits

23. CHOLERA
Causative agent – Vibrio cholerae
Diagnostic Techniques :-
 Culture method
• Gold standard test
• Selective medium is used
• Thiosulphate citrate bile salts
agar
 PCR
 Dipstick Rapid tests
• Not recommended as
confirmatory test
• Result produce in minutes
• E.g. Crystal® VC

24. HEPATITIS
Causative agent – Hepatitis virus (A,B,C,D,E)
Diagnostic Techniques :-
 Blood tests
• Detects antigen & antibodies against
Hepatitis virus
• Used for liver function tests for detection
of -
Albumin, ALP, ALT, AST,
Bilirubin DNA test
• For detection of genetic strand in Hepatitis
B virusAdvanceme
nt- Biopsy
• Liver biopsy
• Tissue sample is taken with the help of long
needle and observed.
 Paracentesis
• Fluid from abdomen is tested
• Obtained with the help of needle
• Helps to identify the severity of
Liver Biopsy

25. MENINGITIS
Causative agent – Neisseria meningitidis, Streptococcus
pneumoniae
Diagnostic Techniques :-
 Blood cultures
• Done with C.S.F
• CSF obtained by lumbar
puncture
• Bacteria identified by using
Gram stain PCR
• To check for antibodies against
certain viruses to determine the
specific cause.
• In case of viral meningitis , CSF
often shows a low sugar level with
increased WBC & increased protein

26. SYPHILIS
Causative agent – Treponema pallidum
Diagnostic Techniques :-
 Serological tests
- 2 types - (both test are done for
diagnosis)
 Non treponemal tests
like
VDRL & RPR
 Treponemal tests like
FTA-ABS, TP-PA,
Immunoblots
 Rapid test
Advanceme
nt-
• Non treponemal test like RPR can also be
considered as rapid test as it provides results in
<10 min.
• Time efficient , cost effective
• Rapid treponemal test also relies on detection of

27. GONORRHOEA
Causative agent – Neisseria gonorrhoeae
Diagnostic Techniques :-
 NAAT (Nucleic acid
amplification test)• World’s most advance tools to detect DNA or
RNA virus
• Uses PCR technology
• DNA probes are usually radiolabelled
• Based on testing for presence of bacteria Advantage -
• Diagnosis of infectious diseases include rapid
detection & identification of infectious agents
• Detection of non viable or difficult to culture
organisms
• May not be useful in low prevalence situation
• Lack of expertise in most diagnostic laboratories
• Expense of reagents used
 Disadvantage
-
 Urine test
 Swab test

28. HIV/AIDS
Diagnostic Techniques :-
 ELISA
• Measures amount of HIV in blood
sample
• Includes RT-PCR, NASBA
 Viral load test
 NAAT
 Western
blot• A confirmatory test for
ELISA
RT-
PCR -
Western
blot

29. DIPHTHERIA
 Schick’s
test
Causative agent – Corynebacterium diphtheriae
Diagnostic Techniques :-
• Diphtheria toxin is injected
into one arm and Heat
inactivated toxin in injected
into another intradermally
• And after 4 days the injected
portion is swallowed and red
if the patient has no
immunity developed against
the toxin.
• And there will be no sign if
immune system responses .

30. Some common diseases & techniques related to their
diagnosis - A close
view

31. A close
view

32. Than