CULTURE ID

2. Identification of bacteria - good isolation must!!
choosing from mixed cultures – challenging
good samples = good report
which is the best sample from above picture and why?

3. PHYSIOLOGY OF MICROORGANISMS
1. Methods of laboratory diagnosis
2. Methods of the bacterial cultivation
3. Identification of bacteria
• Colony characters
• Gram stain
• Motility
• Odor, Pigment
• Biochemical reaction
• Antigen – antisera
• Phage typing
• Antimicrobial resistance or
susceptibility –SPECIFIC abx
• Maldi -TOF
• Genomics
– Typing/sequencing

4. Identification of bacteria
• Motility: Some bacteria can move (Salmonella, E.
coli, Proteus, Pseudomonas, Vibrions, Clostridia).
Dark ground microscopy and Phase contrast
microscopy, special culture media use for
studying motility of bacteria
Special stain for flagella

5. Identification of bacteria
• Culture character: Growth requirement, colonial
characteristics in culture
Colony morphology descriptions

6. Identification of bacteria
• Metabolism: Capacity to form pigment and power of
haemolysis is help for classification of bacteria
Staphylococcus
aureus
Micrococcus
roseus
Studying of
haemolysis

7. Colony morphology

8. Identification of bacteria
• Antigenic analysis: by using specific sera we
can identify microorganism by agglutination
reaction (Serologic Typing of Shigella).
No clumping of the bacteria
is seen in this circle
The clumping of the bacteria
is seen in this circle

9. BIOCHEMICAL
IDENTIFICATION

10. Biochemical tests classified as
1. Single enzyme tests
a. Catalase
b. Coagulase
c. Oxidase
d. Indole
e. DNAase
f. ONPG
g. Urease
h. Hippurate hydrolysis

11. • To differentiate members of the family Microcococcaceae
(including Staphylococcus) which are catalase positive from Streptococcus
species which are catalase negative.
The enzyme catalase catalyzes the release of water and oxygen from
hydrogen peroxide. catalase
Principle: 2 H202  2 H20 + O2 bubbles or effervescence
INTERPRETATION
Positive – rapid and sustained appearance of bubbles or effervescence
Negative – lack of bubble formation 30 seconds later
CATALASE TEST

12. • PURPOSE - To determine the ability of the organism to produce
coagulase which clots plasma.
• To distinguish the pathogenic coagulase positive staphylococcus
from the nonpathogenic coagulase negative staphylococcus.
• Principle: Coagulase is an enzyme that converts soluble fibrinogen
into soluble fibrin.
• Two forms of coagulase
• bound coagulase (clumping factor) – detected in the coagulase slide
test can directly convert fibrinogen to insoluble fibrin and causes the
staphylococci to clump together
• free coagulase – detected in the coagulase tube test reacts with a
globulin plasma factor(coagulase reacting factor-CRF) to form a
thrombinlike factor, staphylothrombin---catalyzes the conversion
• of fibrinogen to insoluble fibrin
COAGULASE TEST

13. INTERPRETATION
Slide Coagulase test
• Positive – white fibrin clots in
plasma
• Negative – smooth
suspension
Tube Coagulase test
• Positive – formation of fibrin
clot
• Negative – no clot is formed

14. OXIDASE TEST
PURPOSE
1.To determine the ability of the organism to hydrolyze the substrate L-
pyrrolidonyl-beta-napthylamide.
2.To differentiate the Enterococcus species from the nonenterococcus
species.
3.Useful for presumptive identification of Group A beta hemolytic
streptococcus(Streptococcus pyogenes)
PRINCIPLE
The cytochrome oxidase test uses certain reagent dyes, such as p-
phenylenediamine dihydrochloride that substitute for oxygen as artificial
electron acceptors
1.It is colorless in the reduced state.
2.In the presence of cytochrome oxidase and atmospheric oxygen, p-
phenylenediamine is oxidized forming indophenol

15. OXIDASE TEST

16. INTERPRETATION
Positive – blue/ dark purple/black color
Negative – no color development
A. Positive – Pseudomonas aeruginosa
B. Negative – Escherichia coli

17. Identification of bacteria
Indole production

18. INDOLE TEST
PURPOSE
•To distinguish Enterobacteriaceae based on the ability to produce indole from
tryptophan.
1.To identify lactose fermenting members of Enterobacteriaceae, Escherichia
coli(indol positive) from Klebsiella pneumoniae(indol negative).
2. To speciate Proteus:
•Proteus mirabilis – indole negative
•Proteus vulgaris – indole positive
PRINCIPLE
•Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing
and deaminating tryptophan with the production of indole, pyruvic acid and
ammonia.
•A red complex is formed when indole reacts with the aldehyde group of p-
dimethylaminobenzaldehyde, the active chemical in Kovac’s and Ehrlich’s
reagent.

19. INDOLE TEST
• Tryptophan  indole+ pyruvic acid + NH3 tryptophanase
• Indole+ p-dimethylaminobenzaldehyde  red complex
Reagents used to detect indole
• Ehrlich’s – to detect indol in anaerobic and nonfermentative bacteria
• Kovac’s – to identify members of Enterobacteriaceae

20. INDOLE TEST
INTERPRETATION
• Positive – red ring at the interface of reagent and broth (or reagent
and xylene or chloroform)
• Negative – no color development
Indole test - EXAMPLES
• A. Positive – Escherichia coli
• B. Negative – Klebsiella pneumoniae

21. UREASE TEST
PURPOSE
• To determine the ability of an organism to produce the enzyme,
urease, which hydrolyzes urea.
• To identify the rapid urease producers(Proteus and Morganella) and
weak urease producers(Klebsiella pneumoniae and species of
Enterobacter)
PRINCIPLE - Urease splits the urea molecule into ammonia(NH3),
CO2 and water(H20).
• Ammonia reacts in solution to form an alkaline compound,
ammonium carbonate, which results in an increased pH of the
medium and a color change in the indicator to pink red.
Urea + 2H2O -------------- CO2 + H2O +2NH3  (NH4)2CO3 - PINK
Urease

22. UREASE TEST
• INTERPRETATION
• Christensens agar
Positive – rapid urease activity; red throughout the medium
Positive – slow urease activity: red in slant initially gradually converting
the entire tube
Negative – no urease activity; medium remains yellow

23. Biochemical tests
2. Assays for metabolic pathways
•Carbohydrate fermentation and Assimilation
a.oxidation fermentation tests
b.carbohydrate fermentation in TSIA
c.methyl red test
d.Voges Proskauer test
•Amino acid degradation
1.decarboxylase-dihydrolase reactions
2.deamination reactions
3.decarboxylation and deamination reactions in LIA
•Single substrate utilization
1.citrate utilization test
2.acetate utilization test
3.acetamide utilization test

24. Identification of bacteria
• Biochemical reactions: The more important
and widely used tests are as under:
Sugar fermentation

25. TRIPLE SUGAR IRON AGAR
PURPOSE: As an initial step in the identification of Enterobacteriaceae
PRINCIPLE:
1. The action of many species of microorganisms on a carbohydrate
substrate results in the acidification of the medium with or without gas
formation.
2. Iron salts(ferrous sulfate and ferric ammonium citrate) reacts with
H2S to produce an insoluble black precipitate(ferrous sulfide).

26. TRIPLE SUGAR IRON AGAR
COMPOSITION
1. Protein sources – beef extract, peptone, yeast extract, proteose peptone
2.Sugars(lactose, sucrose, glucose)
3.Indicators - phenol red – carbohydrate fermentation, ferrous sulfate –
hydrogen sulfide production
4.Sodium thiosulfate – source of sulfur atoms
5.Sodium chloride – osmotic stabilizer

27. BIOCHEMICAL REACTIONS –TRIPLE SUGAR IRON AGAR
carbohydrate fermentation acid production
• yellow deep – glucose fermented
• yellow slant – lactose and/ or sucrose fermented
gas formation bubble formation cracking or splitting of the agar
• upward displacement of the agar
• pulling away of the medium from the walls of test tube
H2S production blackening of the butt(FeS – black precipitate)

33. CITRATE UTILISATION

34. • This test is among a suite of IMViC Tests (Indole, Methyl-Red, Vogues-Proskauer,
and Citrate) that are used to differentiate among the Gram-Negative bacilli in the
family Enterobacteriaceae.
• Media used in Citrate Utilization Test Simmon’s Citrate Agar
• Principle of Citrate Utilization Test
• Citrate agar is used to test an organism’s ability to utilize citrate as a source of
energy. The medium contains citrate as the sole carbon source and inorganic
ammonium salts (NH4H2PO4) as the sole source of nitrogen.
• Bacteria that can grow on this medium produce an enzyme, citrate-permease,
capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s
metabolic cycle for the production of energy. Growth is indicative of utilization
of citrate, an intermediate metabolite in the Krebs cycle.
• When the bacteria metabolize citrate, the ammonium salts are broken down
to ammonia, which increases alkalinity. The shift in pH turns the bromthymol
blue indicator in the medium from green to blue above pH 7.6.
CITRATE UTILISATION

35. CITRATE UTILISATION
• Positive Reaction: Growth with color change from green to intense blue along the
slant.
Examples: Salmonella, Edwardsiella, Citrobacter, Klebsiella, Enterobacter,
Serratia, Providencia, etc.
• Negative Reaction: No growth and No color change; Slant remains green.
Examples: Escherichia, Shigella, Morganella, Yersinia etc.

36. Biochemical tests
• Establishing Inhibitor Profiles
1.bacitracin susceptibility test
2.bacitracin and sulfamethoxazole-
trimethoprim susceptibility test
3.novobiocin susceptibility test
4.vancomycin susceptibility test
5.antibiotic disks for presumptive
identification of anaerobes

37. Other more specific tests
• growth in various NaCl concentrations –
Enterococci and Vibrio species
• susceptibility to optochin and solubility in
bile –Streptococcus pneumoniae
• ability to hydrolyze esculin in the presence
of bile – Enterococcus spp.and Group D
streptococcus
• CAMP – Streptococcus agalactiae

39. API-20 "Bio Merieux" (France) strip test
• Twenty tests are performed on this strip by a
simple procedure, saving time and money.
Escherichia
coli
Enterobacter
agglomerans
Edwardsiella
hoshinae

40. Automated ID & AST

41. Newer ID method

42. THANK YOU