Introduction And History of Microbiology, Subdivision of Microbiology, Classification, Isolation, Isolation techniques, Stains, Types of Staining,

1. Fundamental Principal
of Microbiology
Presented By
Ms. Monika P. Maske
M. Pharm
(Pharmaceutical Chemistry)

2. Introduction
• Microbiology is the study of living organisms that are microscopic in size.
• Microbiology can be defined as the study of living organisms of microscopic
size which include bacteria, fungi, algae, protozoa and viruses.
• It also define as branch of science which deals with the study of
identification, structure, physiology, metabolism and reproduction of mo.

3. • Microorganisms are a heterogenous group of several distinct diseases of living
beings.
• Bacteria belong to a group of living organisms known s microbes.
• Some microbes are beneficial & others are harmful to living beings.
• The term ‘microbe’ taken from French an it means microscopic or micro
organism.

4. History
• Robert Koch is father of bacteriological technique.
• Antoni van Leeuwenhoek is father of Microbiology.
• Robert Hooke first discovered microorganism.
• Mother of microbiology is the Fanny Hesse she developed agar for cell
culture.

5. Subdivision of Microbiology
• Bacteriology – it deals with bacteria
• Mycology – it deals with fungi
• Phycology – it deals with algae
• Protozoology – it deals with protozoa
• Helminthology – it deals with worms
• virology – studies about viruses

6. Classification

7. Basic Classification of Microorganism
Eukaryotes
• Large in size
• Mitochondria present
• Membrane bound nucleus
• Contains all enzymes for production
of metabolic energy.
• Ex. Algae, fungi, protozoa
Prokaryotes
• Small in size
• Mitochondria absent
• DNA not separated from cytoplasm
• Contains all enzymes like eukaryotes
• Ex. bacteria

8. Isolation
• Isolation defined as separating completely and obtaining in pure form of particular type of
micro organisms, separating it from its habitat.
• Growth of microbes in or on a laboratory medium known as culture.
• A growth of this type in solid medium which is originated from a single cell or spore is
termed colony.
• A culture which contains only one species of microbe called as pure culture and which
contain several species called as mixed culture.

9. Isolation Techniques
1. Direct transfer techniques-
• Bacteria, yeast may be found in pure culture under natural conditions.
• Such cultures can be directly transferred to suitable medium.
• Pure culture can be obtained and avoided contamination.

10. 2. Single-Cell Isolation Techniques
• In this technique pick out a single cell of the desired type.
• This done by using micromanipulator in combination with a microscope.
• Micromanipulator has a micropipette with a very fine capillary point.
• Micropipette and single cell can moved as desired.
• Single cell picked using micropipette & transferred to nutrient medium.

11. Disadvantage
• Required skilled operator.

12. 3. Streak Plate Technique
• In this method small amount of sample is transferred on a suitable sterile solid
nutrient medium in Petri dish.
• The sample is streaked by a nichrome-wire loop, it provides successive dilutions and
isolated colonies.
• Streaked method done by 3 methods are, square method, four-quadrant and zig-zag
method.

13. a) Square Method
• One loopful suspension is streaked on solid nutrient medium in horizontal
line.
• Plate is rotated and again streaks are made in vertical lines.

14. b) Four-quadrant Method

15. c) Zig-Zag Method
• A loopful of sample is streaked on solid nutrient agar medium in zig-zag
manner

16. 4. Serial Dilution techniques
• A small amount of material, which contains mixture of bacteria is added to
test tube containing sterile medium of known volume.
• 1 ml of liquid bacteria mixture is then diluted to the desired dilution by
transfer through a series of test tube containing volume of sterilized medium.
• A stage reached when higher dilutions will contains no organisms & show no
growth upon incubation

18. 5. Pour Plate Technique

19. 6. Animal Inoculation Technique
• Some pathogenic bacteria which causes diseases in man & animal do not grow on
artificial medium.
• It may injected in susceptible animal.
• Desired organism may be isolated from food or some tissue of animal.
• Character can determine by observing colonies on agar media, growth on agar slant,
broth.

21. Stains
• Stains are the organic dyes which used for staining the microorganisms.
• Ex. Crystal violets, methylene blue.
• After isolation of the causative microorganisms staining them properly does
morphological study
Purpose of staining-
• Greater visualization of cells
• For study of their structure
• Differentiate the cells
• Inhibit the growth of organisms can visualised

22. Types of Staining

23. 1. Gram Staining
• Christian gram developed differential staining technique, to differentiate types of bacteria
and their specific structure.
• Hence it called as gram’s staining.
• The gram stain procedure distinguishes by grouping colouring these red or violet between
- Gram positive
- Gram negative

24. Gram Stain Procedure
• Bacteria are taken and carefully placed in dry, clean glass dry slide called as
smear.
• Smear is dried by passing small flam.
• Add crystal violet and let stand for 1 minute.
• Tilt the slide slightly and gently rinse with tap water or distilled water using a
wash bottle.
• Gently flood the smear with Gram’s iodine and let stand for 1 minute.

25. • Tilt the slide slightly and gently rinse with tap water or acetone.
• The smear observed under microscope.
Interpretation
Gram Positive: Blue/Purple Color
Gram Negative: Red Color

27. 2. Acid Fast Staining/ Ziehl - Neelsen’s
Staining
• Make a smear. Air Dry. Heat Fix.
• Flood smear with Carbol Fuchsin stain and allowed to react with for 10 min.
• Cover flooded smear with filter paper.
• Steam for 10 minutes.
• Add more Carbol Fuchsin stain as needed.

28. • Cool slide and rinse with Distilled water
• Add 20 % sulphuric acid and leave for 1 min. and washed with water.
• Add methylene blue or malachite green for 30 sec.
• Wash the slide & dry observed under oil immersion lens.

29. Observation:
• Cells appears pinkish red are acid fast cells/ bacteria.
• Cells appears blue/ green are non acid fast bacteria.

31. 3. Endospore
Staining

32. 4. Volutin Granule
Staining Technique

33. 5. Capsule Staining
Technique