MICRO

1. Presented By:
Samjhana Joshi
Kareena Shrestha
Mahanand Yadav
Saju Pun
Suruchi Thapa
Presented To:
Tapeshwar Yadav
Department of
Laboratory Medicine,
Nobel College of Medical
Sciences, Kathmandu,
Nepal

2. LABORATORY
 Laboratory is the place where all the equipments, instruments and
chemical (reagents) are available for performing investigative
procedures, experimental works and research activities by using
biological specimens (whole blood , serum , plasma , urine , stool etc. )
 There are various departments of the clinical laboratory -:
 Clinical chemistry
 Haematology
 Microbiology
 Blood bank
 Support services
( Phlebotomy / Specimen processing)
 Urinalysis
 Immunology
 Serology
 Histopathology and Molecular biology
etc.

3. Microbiology
 Microbiology is the study of
living organisms of microscopic
size which includes bacteria ,
Fungi , Algae , Protozoa and Viruses. It is concerned with the forms,
structure , reproduction , physiology , metabolism and classification.
Principle Of Microbiology
 Medical microbiology deals with the causative agent of the infectious
disease of the human , the ways in which they produce disease in the
body and essential information for diagnosis and treatment.

4. Scope Of Microbiology

5. History of Microbiology
 Long ago , the diseases or the death of
human beings were believed to be punishment
sent from god for human wrong doing.
 Some of the scientists who involved in the development of microbiology
are listed below :
 Robert Hooke divised compound
microscope and looked at the slices of cork .
 Micrographia was an accurate and detailed record of Hooke’s
observation illustrated with magnificient drawing.
 Antony Von Leuweenhoek was the first to observe and describe
the microorganisms on his own made microscope.
 After his invention of microscope, there are number of significant
development. Thus he is known as Father of Microbiology.

6. Golden Era of Microbiology
 During this period we see the real beginning of Microbiology as a discipline of
biology which began with the work of Louis Pasteur and Robert Koch.
 In 1856, Louis Pasteur introduced fermentation techniques , sterilization
techniques that is (Steam Sterilization-1000 C , Autoclave, Hot air Oven-1600 )
and Pasteurization technique.
 There is number of discoveries in his period. Thus,
he is renowned as Father of Modern Microbiology.
 Robert Koch discovered Anthrax bacillus,
Tubercle bacilli and causative agent of Cholera.
 He also introduced the practice of using laboratory
animals for the experiments as he formulated
Koch’s Postulates .
 He also developed pure culture techniques.
Therefore, he is known as a
Father of Modern Bacteriology.
Koch’s Postulates

7. Various Field of Medical Microbiology
 Medical Microbiology
1. Bacteriology
2. Virology
3. Parasitology
4. Mycology
5. Immunology

8. BACTERIOLOGY

9. 1. Bacteriology
The subject Microbiology, is hugely
dependent in this field. Simply, the
study of bacteria is called as bacteriology.
Generally, size of bacteria is about 0.2-1.5µm
in diameter. Bacteria consists of both DNA and RNA in the
form of nucleiod in the nuclear material of it.
CLASSIFICATION OF BACTERIA
Bacteria can be classified int0 various types they are -:

10.  On the basis of Morphology
A .True Bacteria
• Cocci – They are spherical or oval shape on the basis of arrangement
of individual organisms they can be descibed as -:
a) Monococci(cocci in singles)(e.g. Monococcus spp.)
b) Diplococci are pairs of cocci (e.g. Streptococcus
pneumoniae and Neisseria gonorrhoeae)
c) Streptococci are chains of cocci (e.g. Streptococcus pyogenes).
d) Staphylococci are irregular (grape-like) clusters of cocci
(e.g. Staphylococcus aureus).
e) Tetrads are clusters of four cocci arranged within the same plane
(e.g. Micrococcus sp.).
f) Sarcina is a genus of bacteria that are found in cuboidal
arrangements of eight cocci.

12.  Bacilli – Bacilli are the rod shaped bacteria divided into various
types on the basis of arrangements.
I. Diplobacilli
II. Streptobacilli
III. Pallisades
IV. Chinese letter form
V. Coccobacilli
VI. Comma shaped (vibrio)
B. Actinomycetes (actin- ray , mukes – fungus ) – They are like
true bacteria but they resemble fungi in that they exhibit
branching X- form filaments.
C. Spirochetes – They are longer, non branched spiral shaped
microorganisms having several coils. Eg- Treponema,
Leptospira etc.

13. D. Mycoplasm – these are bacteria lacking cell wall and
highly pleomorphic in nature. Eg . L- form,
Spheroplast , protoplast.
E. Rickettsiae and Chalamydiae - Very small bacteria
which are obligatory in nature.
 On the basis of Anatomical features
Capsule –
Capsulated – Streptococcus pneumoniae
Non-capsulated – Streptococcus viridans

14. Flagella –
 Aflagellate (eg. Shigella spp. )
 Monotrichous (Single flagella i.e Vibrio )
 Lophotrichous (Group of flagella at one end
i.e Helicobacter )
 Amphitrichous (Single flagella at both end)
 Peritrichous ( Flagella all around body i.e
Salmonella , Escherichia coli )
Spores
 Spore forming – Bacillus spp.
 Non spore forming – Escherichia coli

15.  Based on Staining
Gram’s Stain
Acid Fast Stain
- Acid Fast bacilli ( Mycobacterium tuberculosis )
- Non – Acid Fast bacilli ( Staphylococcus aureus )

17. STERILIZATION AND DISINFECTION
Sterilization
It is the process by which medium is freed of all living microorganism either in vegetative
form or spore state.
Disinfection
Substance interfare with membrane function. Eg; Phenol, chloroform, H2O2, Formaldehyde
etc.
Protein denaturing agent : HCL (modify functional group of protein) .
Sterilization Agents
 HEAT (Most reliable method of sterilization)
A)Dry heat:
 It denaturate the protein of bacteria and
cause oxidative damage.
 It is done by flaming (needle, cotton wool),
red heat(Inoculating loop), Incineration
(Pathological materials ,solid dressing),
Hot air oven (Glasswares) etc.

18. B)Moist heat
Temperature below 100˚c
 Vaccine bath- 56˚c for 1 hour for 3 successive day.
 Pasteurization of milk.
- 63˚c for 30 min (Holder Method)
- 72 ˚c for 15 sec (Flash Method)
- Inspirator at 80 ˚ -85 ˚C
Temperature at 100˚c
 Tyndallization (3 successive days ) eg: for media
 containing sugar or gelatin.
Temperature above 100˚c
Steam under pressure- Autoclave for dressing
instruments , media , laboratory wares etc under the
temperature of 121 ˚ c for 15-20 min at 15 lbs pressure.

19. C) Filtration : Other method of Sterilization for the heat labile liquid for examples :
Serum,toxin , glucose solution. Filters like sintered glass filter, membrane filter etc.
D) Radiation: Non ionization (IR ray) – Syringes
UV ray – Operating theatres
and Laboratories.
Ionization – (X-ray,Gamma ray)- For plastic
syringes,Catheters, rubber
goods etc.
E)Gas Vapour Sterilization :
 Ethylene Oxide – Plastic goods,culture medias
 Aldehyde – Operation Theatre
 Hydrogen Peroxide – For contact lense
 Halogens – as laboratory disinfectant on surface
of bench and in discard spots.
 Alcohols - 70% alcohol is used
 Phenols - Lysol for disinfection of excreta,
Floor of Ward and operation room.

20.  Laboratory Procedure used in Bacterial
Disease Diagnosis :
1. Microbial Culture
It is primary method for isolation of
infectious bacteria or diseases in the
laboratory.
These isolated bacterias can also be used for
studying its size , structure, morphology , for researches and other
various purposes. Culture is done in laboratory by various
methods like:
 Streak culture
 Stroke culture
 Carpet culture
 Stab Culture
 Sweep Plate method

21. Carpet Culture Streak Culture

22.  There are 3 types of media which are used for the growth of
bacteria using the culture methods.
 Solid Culture Media – It contains 1.5-2%
of agar base. This media is primarily
used to culture bacteria. Some of the
examples of this media are Basal media ,
Enriched media , Differential media ,
Indicator media, Selective media
Transport media etc.
 Liquid Culture Media – This media is used for the growth of the cells
inside its liquid media (used for detecting Mycobacteria). Eg.
Enrichment media ( Thioglycollate broth , Nutrient broth ) etc.
 Cell Culture – This method is used to determine the microbe on the
cells for the identification of viruses.
.

24. 2. Microscopy
Culture techniques will often use a
microscopic examination to help in the
identification of microbes or bacteria.
Various types of microscope are used
for this purpose some of them are
i. Compound microscope
ii. Electron microscope
iii. Light microscope
iv. Fluorescence microscope
v. Dark field microscope.

25. Staining Methods
 Gram’s Stain
Danish scientist Hans Christian Gram devised a method to
differentiate two types of bacteria based on the structural
differences in their cell walls. In his test, bacteria that
retain the crystal violet dye do so because of a thick layer of
peptidoglycan and are called Gram-positive bacteria. In
contrast, Gram-negative bacteria do not retain the violet
dye and are colored red or pink. Compared with Gram-
positive bacteria, Gram-negative bacteria are more
resistant against antibodies because of their impenetrable
cell wall. These bacteria have a wide variety of applications
ranging from medical treatment to industrial use.

27. AFB / Ziehl Neelsen Stain
It is used to detect the presence of
acid fast bacilli like
Mycobacterium tuberculosis &
Mycobacterium leprae.
Interpretation
Acid Fast Bacilli – Red or Pink in colour
Background – Blue or Green (according to counterstain used)
 Other special Stains
- Capsule : India Ink
- Spore : Moeller stain

28. Biochemical Tests
Fast and relatively simple biochemical test can be used
to identify infectious agents. For bacterial identification,
the use of metabolic or enzymatic characteristics are
common. Acids, alcohol and gases are usually detected
in this tests.
 Biochemical tests performed are :
Catalase test
Coagulase test
Indole test
Bile solubility test
Oxidase test
Urease test

29. Serological Method
Highly sensitive and specific method which is based upon
the ability of an antibody to bind specifically to an antigen.
PCR (Polymerase Chain Reaction)
For detection and study of microbes. Which is definite
accurate and fast.

30. Medical microbiologists often make treatment recommendations to the patients physician based on
the strain of microbes and its antibiotic resistance , the toxicity of antimicrobial drugs and drug
allergies the patient has by performing AST.
For performing AST, modified Kirby-Bauer method is recommended and Mueller Hinton Agar is used.

31. Virology
It is the study of Viruses which are obligate
intracellular parasites, size of viruses is about
20-400 nm.
Largest Virus – Pox Virus
Smallest Virus – Picorna Virus
Viruses either contain DNA or RNA but never both
Cancer causing (Oncogenic Virus )
 Hepatitis C Virus
 Hepatitis B Virus
 Human Pappiloma Virus
RNA Viruses DNA Viruses
Retro viruses, Rhino Viruses Parvo Virus , Pox Virus
Hepatitis A,C, D,E Hepatitis B Virus
Rota Virus Adeno Virus

32. Laboratory Procedures Used in Viral Disease Diagnosis :
1. Specimen Collection : Specimen such as Throat swab ,
Nasal swab , Bronchial washing, Urine , Stool , Rectal swab and
CSF etc are used for detection.

33. 2. Viral Transport Medium : It is used for transportation of
Viruses from one place to another.
3. Diagnostic Method in Virology :
i. Direct examination : By Ag Detection, Electro
microscopy , Light microscopy , Viral genome detection
etc.
ii. Indirect examination : By cell culture, egg inoculation
and animal inoculation.
iii. Serology test : They are Ab detection neutralization test ,
ELISA, Western blot etc.

34. PARASITOLOGY
Parasitology is the area of
biology concerned with the
phenomenon of dependence of
one living organism on another.
Medical parasitology deals with the
protozoal as well as helminthic parasites, which infect different
organ systems of human beings, the disease they produce,
various methods of diagnosis and prevention. In lab, smear
preparation for microscopy, cultures and serology test is done
for detection of parasites.

35. MYCOLOGY
It include the study of fungus and fungal infection. Fungus causes
the eczema and allergy.
The disease cause by fungal infection is called Mycotic disease.
Fungus may infect nail, hair, skin and organs. Fungi is the causative
agent of ringworm and tinea.
INFECTION OF FUNGI
A. Superficial Infection:
Infection in skin, hair, nail, eye, ear etc by Microsporum,
Trichopytous and Epidermophyton etc.
A. Cutaneous Infection: Infect on cutaneous layer which is caused
by saprophytic fungi.
B. Systemic Infection: Fungal Infection in various organs caused by
Histoplasm Capsulatum , Blastomyces Dermatidis etc.

36. LABORATORY DIAGNOSIS OF FUNGAL
DISEASE
1. Microscopy :
a. 10 % KOH preparation
b. India Ink
c. Stain – Giemsa , PAS stain
2. Culture :
Commonly used Media is SDA ( Sabouraud Dextrose Agar )
BSA ( Bird Seed Agar for Cryptococcus )

37. IMMUNOLOGY
The term ‘ immunity’ is defined as resistance
exhibited by the host against any foreign
antigen including microorganisms.
This resistance plays a major role in
prevention of infectious diseases .
Antigen : It is a substance which , when introduced into a body
evokes immune response to produce a specific antibody with which
it reacts in an observable manner.
Antibodies: It is a substance which are formed in the serum and
tissue fluids in response to an antigen and react with that antigen
specifically and in some observable manner.
Antigen- Antibody Reaction
Ag-Ab reaction is reversible , occurs at surface and there is no
denaturation of Ag and Ab during reaction.

38. Types of Ag – Ab Reaction
1. Precipitation Reaction : More sensitive for antigen detection. Eg:
Biken Test.
2. Flocculation Reaction : Special types of precipitation where
precipitation remains suspended instead of sedimentation Eg.
VDRL, RPR .
3. Agglutination Reaction : More sensitive than precipitation for
antibody detection. Antigen – antibody reaction in which
antibody combine with a particulate antigen for eg. Widal Test ,
Sero-typing etc.
4. Neutralisation Test : When anti toxins combines with its
corresponding toxin , neutralization occurs. Eg; Schick Test,
Dick Test.
5. Radio Immuno Assay (RIA) : It is used for quantification of
hormones , drugs, tumor markers , viral antigen etc.
6. Enzyme Immuno Assay (EIA) : It measures enzyme labelled
antigen or antibody. Eg; ELISA, RF (Rheumatoid Factor).

39. Biological Safety Cabinet(BSC)
BSC is an enclosed, ventilated laboratory works place for
safety working with materials contaminated with pathogens
requiring a defined biosafety level. It protect a laboratory
worker and environment from aerosols and air borne particles.
Biosafety Cabinet Classification
Types of cabinet Agent classification
(Biosafety level)
Protection
Class I Low and moderate risk User only
Class II Low and moderate risk User and materials
Class III High risk User and environment

41. Quality Assurance (QA)

43. THANK YOU