Fundamental Principal of Microbiology

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
•Microbiologyisthestudyoflivingorganismsthataremicroscopicinsize.
•Microbiologycanbedefinedasthestudyoflivingorganismsofmicroscopic
sizewhichincludebacteria,fungi,algae,protozoaandviruses.
•Italsodefineasbranchofsciencewhichdealswiththestudyof
identification,structure,physiology,metabolismandreproductionofmo.

3. •Microorganismsare 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 Frenchan it means microscopic or micro
organism.

4. History
•Robert Koch is fatherof bacteriological technique.
•Antoni van Leeuwenhoek is fatherof Microbiology.
•Robert Hooke first discovered microorganism.
•Motherof 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
•Asmallamountofmaterial,whichcontainsmixtureofbacteriaisaddedto
testtubecontainingsterilemediumofknownvolume.
•1mlofliquidbacteriamixtureisthendilutedtothedesireddilutionby
transferthroughaseriesoftesttubecontainingvolumeofsterilizedmedium.
•Astagereachedwhenhigherdilutionswillcontainsnoorganisms&showno
growthuponincubation

18. 5. Pour Plate Technique

19. 6. Animal Inoculation Technique
•Somepathogenicbacteriawhichcausesdiseasesinman&animaldonotgrowon
artificialmedium.
•Itmayinjectedinsusceptibleanimal.
•Desiredorganismmaybeisolatedfromfoodorsometissueofanimal.
•Charactercandeterminebyobservingcoloniesonagarmedia,growthonagarslant,
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
•Bacteriaaretakenandcarefullyplacedindry,cleanglassdryslidecalledas
smear.
•Smearisdriedbypassingsmallflam.
•Addcrystalvioletandletstandfor1minute.
•Tilttheslideslightlyandgentlyrinsewithtapwaterordistilledwaterusinga
washbottle.
•GentlyfloodthesmearwithGram’siodineandletstandfor1minute.

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

27. 2. Acid Fast Staining/ Ziehl-Neelsen’s
Staining
•Makeasmear.AirDry.HeatFix.
•FloodsmearwithCarbolFuchsinstainandallowedtoreactwithfor10min.
•Coverfloodedsmearwithfilterpaper.
•Steamfor10minutes.
•AddmoreCarbolFuchsinstainasneeded.

28. •CoolslideandrinsewithDistilledwater
•Add20%sulphuricacidandleavefor1min.andwashedwithwater.
•Addmethyleneblueormalachitegreenfor30sec.
•Washtheslide&dryobservedunderoilimmersionlens.

29. Observation:
•Cellsappearspinkishredareacidfastcells/bacteria.
•Cellsappearsblue/greenarenonacidfastbacteria.

31. 3. Endospore
Staining

32. 4. Volutin Granule
Staining Technique

33. 5. Capsule Staining
Technique