Identification of Bacteria

Identification of bacteria, Staning reagent, Biochemical test

1. MS. FAND S. B.
ASSISTANT PROFESSOR ACOP, AHMEDNAGAR
IDENTIFICATION OF BACTERIA

2. CONTENTS
oINTRODUCTION
oSTAINING REACTIONS
oBIOCHEMICAL TESTS

3. INTRODUCTION
✓Identificationistheprocessofdeterminingtoestablishedtaxonanewisolateorunknownstrainbelongs.
✓Identificationofunknownbacterialcultureisoneofthemajorresponsibilitiesofamicrobiologist.
✓Samplesofblood,tissue,water,foodandcosmeticsareexamineddailyinlaboratoriesthroughouttheworldforthepresenceof
contaminantsandpathogenicmicroorganisms.
✓Pharmaceuticalindustriesandresearchinstitutesareconstantlyscreeningsoil,water,marinesampletoisolatenewantibiotic,
enzymes,andvitaminproducingmicroorganism.
✓Thescienceofclassificationiscalledtaxonomy.

4. INTRODUCTION
✓Bergey'sManualofSystemicBacteriology(BMSB)hasbeentheofficial,internationallyacceptedreferenceforbacterial
classification.
✓InthecurrenteditionofBergey'smanual,bacteriaisclassifiedinto33groupscalledsectionsandcontaininformationof4000
bacterialspeciescalledsectionsratherthanintotheclassicaltaxonomicgroupingsofphylum,class,orderandfamily.
✓Therelationshipofmicroorganismsineachsectionisbasedoncharacteristicssuchasmorphologystainingreactions,
nutritionalrequirements,physiologicalproperties.
✓Culturalcharacteristics,biochemicaltests,serologicalproperties,pathogenicityandgeneticcharacterization.

5. STAINING TECHNIQUE
Cell Shape
Cell Size
Cell Arrangement
Contrast Differential Stain Procedure
Simple stain
technique
Negative stain
technique
Gram
staining
Acid-fast
staining
Basic Dye Basic Dye
Gram -ve
staining
Gram +ve
staining
Blue/ Purple cells Orange/ Red cells

6. STAINING TECHNIQUE
✓To Study Size, Shape, Arrangement And Properties And
Different Specific Groups Of Microorganisms, Biological Stains
Are Used.
✓Stains Is An Organic Compounds Containing A Benzine Rings
With Chromophore And Auxochrome Group.
✓Different Types Of Staining Techniques Are Used To Study The
Morphological And Structural Properties Of Microorganisms.

7. DYES
oMethylene blue, crystal violet, safranin,
basic fuchsin etc.
oPositively charge group
oGenerally chloride salts
oBasic dye bind to positively charged
molecules nucleic acid, proteins, surface
of bacterial cell.
oUsed for positive staining.
oEosin, Rose Bengal, Nigrosine, Congo
red, Acid Fuchsin (Indian Ink)
oSuch as carboxyl's (-COOH), phenolic
hydroxyls (-OH)
oAcidic dyes ionized form negative charge
& bind to positively charged cell
structure
oUsed for negative staining.
Basic Dye Acidic Dye

8. In the staining technique, bacterial
culture is fixed on the slide with
heat by flaming underneath slide.
Fixed smear is stained by various
following staining technique.
1.Simple staining
2.Negative staining
3.Impregnation method
4.Differential staining

9. SIMPLE STAINING
✓In simple staining, the bacterial smear is stained with a single stain e.g. methylene blue Crystal violet, carbol fuchsin safranin
etc.
✓ Basic stains with a positively charged chromogen are used.
✓Bacterial nucleic acids and certain cell wall components carry a negative charge that strongly binds to the cationic chromogen.
✓The purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells.

10. PROCEDURE
1
•A clean grease free slide is taken A grease free slide is made by first washing the
slide with detergent wiping the excess water and the slide is passed through flame.
2
•On these grease free slide smear is made by using a sterile wire loop and cell
suspension.
3
• These slide is allowed to air dry.
4
•After air drying these slide is rapidly passed through a flame for three to four times
for heat fixation.
5
•After heat fixation the slide is placed on the staining rack and flooded with a
particular stain and this stain is allowed to react for three minutes.
6
•Further the slide is washed under running water.
7.
•The slide is air dried and observed under microscope.

11. •The surface of a bacterial cell has acidic characteristic because of a large amount of carboxyl groups located
on the cell surface due to acidic amino acids.
•when ionisation of carboxyl groups takes place, it imparts negative charge to the cell surface.
•In nature, H is replaced by another positive charged ion, e.g. Na+ or K+ and H+ bonds with oxygen to form
water. Thus, surface of an unstained bacterial cell is represented as
•Basic dyes are commonly used for the monochrome staining.
MECHANISM
Na+
Na+
Na+Na+
Na+
Na+
Na+Na+ MB+
MB+
MB+MB+
MB+
MB+
Na+MB+
Methylene blue
chloride (MB+ Cl
-
)
+ NaCl
COOH COO
-
+H+
Ionisation

12. •These dyes are available as a salt of acids. e.g. methylene blue chloride.
•When methylene blue rehydrates, it ionizes to form methylene blue and chloride ions. The positively
charged ions have the colouring property.
Methylene blue chloride Methylene blue Chloride
•On addition of methylene blue for staining, exchange of MB+ with Na+ on the bacterial cell surface takes
place, resulting into lonic bond formation between MB and cell surface.
•Thus, when colouring agent forms ionic bond with cell or cell components, it results into the staining of cell.
The most commonly used basic stains are methelyne blue (2 to 3 minutes), crystal violet (1 to 2 minutes)
and carbol fuchsin (15 to 30 seconds)
MECHANISM
MB. Cl MB+ +Cl
-
Ionisation

13. AdvantageofSimpleStaining:
•Simplestainingisareasonablysimpleprocedurethatjustrequiresonereagenttostaintheorganism.
•Itisaquicktechniquethattakesonly3-5minutestocompletetheperformance.
•Simplestaininghelpsinexaminingorelucidatingtheshape,size,andarrangementofthemicroorganisms.
•Italsoenablesustodistinguishbetweenbacterialcellsandinanimateobjects.
•Simplestainingmightbehelpfulinthepreliminaryanalysisofthemorphologicalcharactersofthebacteria.
SIMPLE STAINING

14. NEGATIVE STAINING
•In negative staining, bacteria are mixed with acidic stain (eosin or nigrosin) and a smear is prepared.
•The acidic stain (negative chromogen) does not penetrate the cells because of the negative charge on the surface of
bacteria. Hence, unstained cells are easily observed against the coloured background.
•This technique is also useful in demonstration of bacterial capsule.
•Negative (indirect) staining is a technique by which bacterial cells are not stained but are made visible against
dark background. The acidic stain is used in this staining e.g. eosin, nigrosin, congo red, Rose Bengal stain etc.
•Acidic stain has negative charge, therefore, it does not combine with negatively charged of bacterial cell surface.
•On the other hand, it forms a deposit around the cell, resulting in appearance of bacterial cell colouriess against
the dark background.

15. AdvantageofNegativeStaining:
•Directorpositivestainingforthestudyofmorphologyofcells.
•Heatfixationisnotrequiredforthecellsthatdonotreceivevigorousphysical&chemicaltreatments.
•Naturalsizeandshapeofmicroorganismscanbeseenbythismethod.
NEGATIVE STAINING

16. GRAM STAINING
•The most commonly used differential stain is the Gram stain, first described in 1884 by Christian Gram.
•The Gram stains divides bacteria into two groups, i.e. Gram-positive and Gram-negative.
•Those organisms which retain the primary stain (crystal violet) are stained purple and are designated Gram-
positive; those which lose the crystal violet and safranin counterstain appear red and are designated Gram-
negative.
•Method consist four components:
1.Primary stain : Crystal violet
2.Mordant : Iodine solution
3.Decolorizer agent : Alcohol/Acetone
4.Counter stain : Safranin

17. 1.Primarystain(Crystalviolet):
•First,crystalviolet,aprimarystain,isappliedtoaheat-fixedsmear,givingallofthecellsapurplecolor.
2.Mordant(Gram’siodine):
•Definedas“anysubstancethatformsaninsolublecompoundwithstainandtofixthecolourtobacterial
cell”.
•Itshouldthatmordantisnotastain.
•ItleadscomplexinGram+vebacteriaCrystalVioletIodine-Magnesiumribonucleate(CV-I-Mg
ribonucleate)
•ThiscomplexnotformedinGram–vebacteriaasMg-ribonucleateabsentincellwall.HenceCV-Icomplex
formedinGram–vebacteria.
GRAM STAINING

18. GRAM STAINING

19. •The Ziehl-Neelsen stain, also known as the acid-fast stain described by Franz Ziehl (1859 to 1926), a
bacteriologist and Friedrich Neelsen (1854 to 1894) a pathologist
•Mycobacterial cell walls contain a waxy substance composed of mycolic acids.
•These are ẞ-hydroxy carboxylic acids with chain lengths of up to 90 carbon atoms.
•The property of acid fastness is related to the carbon chain length of the mycolic acid found in any particular
species.
•This staining technique divides bacteria into two groups namely acid-fast and non acid-fast.
ACID-FAST STAINING

20. ACID-FAST STAINING

21. ACID-FAST STAINING
Procedure:

22. ACID-FAST STAINING

23. ACID-FAST STAINING

24. ACID-FAST STAINING

25. ACID-FAST STAINING

26. ACID-FAST STAINING

27. ACID-FAST STAINING

29. INDOLE TEST

30. INDOLE TEST

31. INDOLE TEST

32. METHYL RED TEST

33. METHYL RED TEST
CO2 + H2
(pH 4.0)
Lactic acid
Acetic acid
Formic acid
(Organic acid)
Glucose + H2O
Red colour
(+ve Reaction)
E. coli
Methyl red
indicator
Ethanol + Acetyl
methyl carbinol
CO2 + H2
(pH 6.0)
Acetic acid Glucose + H2O
Yellow colour
(-ve Reaction)
E. aerogenes

34. VOGES-PROSKAUER TEST

35. VOGES-PROSKAUER TEST

36. VOGES-PROSKAUER TEST

37. CITRATE UTILIZATION TEST
1. Sodium Citrate:

38. CITRATE UTILIZATION TEST

39. CITRATE UTILIZATION TEST
2. Urease Test:
•Urease is an enzyme produced by certain micro- organisms which converts the urea to ammonia & CO2.
•Development of ammonia will increase the PH of the media and converts colorless phenolphthalein to
pink color.
Requirements:
1.Christensen's urease medium.
Procedure:
Inoculate the test organism from pure culture in to Christensen's medium, incubate at 35°C for 18 to 24hrs.

40. CITRATE UTILIZATION TEST
3. Nitrate Reduction:
Principle:
•The capability of an organism to reduce nitrates(NO3) - nitrites (NO2) will be detected.
•The organisms which has got enzyme known as "Nitrate reductase", have the capability of extracting
O₂ from Nitrates to form Nitrites.
Procedure:-
•Inoculate nitrate medium with test organism.
•Incubate at 35°C for 96 hrs and add 1 ml each of a-naphthalamine and sulfanilic acid in 5ml acetic
acid mixed.
•Positive Control:- All enterobactericae, Haemophilus, Neisseria, Moraxella species.
•Negative Control: Acinetobacter baumannii.

41. CITRATE UTILIZATION TEST
4. Sugar fermentation:
•Bacteria produce acidic products when they ferment certain carbohydrates.
•The carbohydrate utilization tests are designed to detect the change in pH.
•Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow.
•If the bacteria do not ferment the carbohydrate then the media remains red or pink.
•If gas is produced as a by product of fermentation, then the Durham tube will have a bubble in it.

42. CITRATE UTILIZATION TEST
Principle:
•To detect the presence of cytochrome oxidase enzyme.
•Some bacteria possess enzyme oxidase that will catalyze the transport of electrons between electron donors
in bacteria and redox dye tetramethyl-P-Phenylene-diamine.
•This dye reduces to a deep purple colour.
Procedures:
3 methods: 1. Plate method 2. Dry filter method 3. Wet filter method
•Moist the filter paper with the substrate of 1% Tetramethyl- P- Phenylamine dihydrochloride (oxidase
reagent).
•Take pure colony to be tested on a wooden stick and rub over filter paper.
•See for purple colour development within 10 seconds.

43. CITRATE UTILIZATION TEST