Practical 40 biochemical test microbiology.pdf

urea breath

1. Prepared By
DR. Hanan Fathy
DR. Sabah Al-Khawagah
Diagnosis of Bacterial
Diseases

2. Intended learning outcomes (ILOs)
Describehowstaining,cultureandbiochemical
testsareusedtoidentifybacteria.
Listthemajorbiochemicaltestsusedtoidentify
bacteria.
Explainhowserologicaltestscanbeusedto
identifyanunknownbacterium.
Toidentifytheimportanceofanimalinoculationin
bacterialidentification.

3. Intended learning outcomes (ILOs)
To understand the principle and uses of bacteriophage
typing.
Toknowtheprincipleandusesofantibioticsensitivity
test.
Todifferentiatebetweendifferentmolecularmethodsfor
bacterialidentification.
Toidentifyphenotypicandgenotypicmethodsusedfor
bacterialtyping.

4. Bacterial identification
Themethodsusedforisolationandidentificationoforganisms
are:
1-Direct microscopic examination of:
✓Freshunstainedfilmtodemonstratethemotilityofthe
organism.
✓Stainedfilme.g.byGramstaintostudyshape(coccior
bacilli…),size(smallorlarge…),arrangement(groupor
chains…)andstainingreaction(Gram+or-ve).

5. Gram negative cocci (pink in
colour)

6. Gram positive bacilli (violet in
colour)

7. Spiral shape bacteria (spirochetes)Gram-negative curved bacilli (V. cholera)

8. Gram positive cocciarranged in
clusters

9. 2-Cultural Morphology:
Thisincludesthecolonymorphology;
▪Size&shape
▪Mucoidordry
▪Opaqueortranslucent
▪Pigmentproduction
▪Theeffectofthecolonyonthesurroundingmedium(e.g.
haemolysisonbloodagarandswarmingonnutrientagar).

10. Pseudomonasculture on nutrient
agar produce greenish exopigment.
Staph culture on nutrient agar
produce endopigment.
1: Staph. Aureus.
2: Staph. albus
3: Staph. citreus
1
2
3

11. Culture of E.colion MacConky
medium (dry rose pink colonies)
Culture of Klebsiellaon MacConky
medium (mucoidrose pink colonies))

12. 1
3
2
Proteus on nutrient agar showing
swarming growth
Blood agar plate showing:
1-Complete hemolysis (Beta)
2-Partial hemolysis (Alpha)
3-No hemolysis (gamma))

13. 3-Biochemical reactions:
•Sugar fermentation
•Triple sugar iron test
•Indoletest
•Methyl red test
•Citrate utilization test
•Catalase test
•Coagulase test
•Urease test
•Oxidase test

14. Sugar fermentation test
▪A set of 5 tubes containing glucose, lactose, maltose, manniteand
sucrose are used.
▪A positive test gives red reaction with or without gas production.

15. Triple sugar iron test (TSI)
▪This test measures sugar fermentation (glucose, lactose and sucrose
and detects H2S production.
▪Positive H2S gives black coloration of the butt.
Un-inoculated

16. Indoletest
▪Thistestdemonstratetheabilityofthebacteriatodecompose
theaminoacidtreptophanpresentinpeptonewaterand
productionofindolewhichistestedbyKovac'sreagent.
▪Positivereactiongivespinkringwhilenegativeoneproduce
yellowring.

17. Methyl red test (MR)
Thistestisdonetodetecttheabilityofthebacteriatoproduce
largeamountofacidonfermentationofglucose.Fewdropsof
MRindicatorareused.
Positivereactiongivesredcolourwhilenegativeonegives
yellowcolour.

18. Citrate utilization test
Theorganismthatabletoutilizethesodiumcitrateasthe
sourceofcarbon,cangrowonthemediawithchangeofits
colourfromthegreentoblue.
Theorganismthatcannotutilizethecitrate,givesnogrowth
andnochangeincolour(green).

19. Catalase test
This test detects the catalase enzyme production by some
baceriae.g. Staphylococci.
It is done by adding a drop of hydrogen peroxide on the test
colony on N. agar.
Rapid effervescence due to oxygen gas production indicates a
positive test.

20. Coagulase test
Coagulasetestisusedtodetectfreecoagulaseenzymeby
addingfewdropsofanovernightbrothcultureofthetest
organismto0.5mlofhumanorrabbitplasmadiluted1/10.
Apositiveresultgivesadistinctclot.

21. Urease test
Some bacteria e.g. Proteus produce urease enzyme which
decomposes urea with the release of amonia. This leads to
alkalinity of medium and change colourof the indicator.
Urease positive bacteria will turn the medium pink after 4-24
hr.

22. Oxidase test
Somebacteriaproduceoxidaseenzymewhichcanreduce
oxidasereagenttoadeeppurplecolour.
Thetestedcolonyispickedandsmearedonastripoffilter
paperimpregnatedwithoxidasereagent.

23. 4-Bacterial motility on soft agar:
▪It is tested by stabbing the bacteria in deep column of soft agar.
▪Non motile organism grows along the stab only.
▪Motile bacterial growth appears radiating from the stab line
and also on the upper surface of agar.

24. 5-Serological tests:
▪Slide agglutination test is used for identification of unknown
colony of bacteria using commercially prepared antisera.

25. 6. Animal pathogenicity test
●Animal inoculation:
Animal commonly used:
-Guinea pigs.
-Mice.
-Rabbits.
●Importance of animal pathogenicity test;
-Identify certain pathogenic bacteria e.g. T.B
bacilli (characteristic lesion).
-Determine degree of pathogenicity of certain
bacteria.
-Differentiate between related organisms.
-Isolation of organism in pure culture.
-Test ability of toxin production.
-Evaluation of vaccine and antibiotics.

26. 7. Bacteriophages typing
●Lysisof bacteria by one or a series of specific bacteriophages.
●Used to trace the source of infection during epidemics e.g. Staph. aureus.

27. Bacteriophagetyping
A number of specific phages leads to lysisof the bacteria.

28. 8. Antibiotic susceptibility test
●Used for selection of the proper antimicrobial agent to be used for treatment.
●Disc diffusion method.

29. Antibiotic sensitivity test (Disc diffusion method)

30. 8. Molecular methods for bacterial identification
●DNA probe:
-Single stranded labeled DNA (probe) hybridize with complementary nucleic
acid sequence in specimen to form double stranded DNA.
●Polymerase chain reaction (PCR):
-Extraction of DNA.
-Amplification of DNA using specific primers in special apparatus.
-The amplified DNA (amplicon) run in gel electrophoresis to it into bands.
●Real-time PCR:
-Automated closed system.
-Rapid and quantitative but expensive.

32. Dot blot hybridization

33. Conventional PCR

35. Agarose gel electrophoresis

36. Polymerase chain Reaction (PCR)

37. 9. Bacterial typing
I-Phenotypic methods:
●Biotyping.
●Antibiotic susceptibility.
●Bacteriophagetyping.
●Serotyping.
II-Genotypic methods:
●Plasmid finger printing.
●Restriction fragment length polymorphism (RFLP).

38. Genotyping
Plasmid finger printing:
•Separation of plasmid DNA from
chromosomal DNA.
•Plasmid DNA is subjected to restriction
enzyme digestion.
•Gel electrophoresis separate the DNA into
a number of bands that are considered as
a fingerprint to each strain.
RFLP Plasmid fingerprint

39. Plasmid Finger Printing