This document discusses methods for identifying bacteria, including conventional culture techniques and automated methods. Conventional culture techniques involve collecting samples, staining, culturing on media, and performing biochemical tests to identify colonies based on morphology, staining characteristics, and biochemical reactions. Automated methods include using blood culture systems to rapidly detect microbial growth and identify organisms using MALDI-TOF mass spectrometry or systems like Vitek-2 that can identify bacteria and perform antibiotic susceptibility testing.

1. IDENTIFICATION OF BACTERIA
Dr amit kumar
amitkumarshahi90@gmail.com

2. IDENTIFICATION OF BACTERIA
• Methods :
• A) Culture and identification
• B) Automated culture techniques
• C) Molecular techniques

3. Microbial identification

4. • A) CONVENTIAL CULTURE METHODS:
• 1)SITE OF INFECTION ( urine) GRAM STAIN 1)BA & MA :PUS , WOUND SWAB,
• 2)PROPER COLLECTION (mid stream urine) ACID FAST STAIN STERILE BODY FLUID ,
• 3)STANDARD PRECAUTION ALBERT STAIN URINE , RESPIRATORY SAMPLES
• 4)COLLECT PRIOR TO ANTIBIOTICS 2) CA: RESPIRATORY AND STERILE BODY FLUID
• 5)AVOID CONTAMINTION 3)DCA , TCBS , XLD: STOOL SAMPLE
• 6)MUST LABELLED 4) BLOOD CULTURE BOTTLE: BLOOD
• 7PROCESSED SOON ( WIHOUT DIRECT MICROSCOPY)
IF DELAY TRANSPORT MEDIUN 5)CLED: URINE
• SIZE : MILIMETER, PIN POINT: STREPTO , PIN HEAD: STAPHYLO
gram&BIOCHEMICALS
SHAPE : CIRCULAR OR IRREGULAR
SURFACE : SMOOTH , ROUGH GRANULAR
• ELEVATION : FLAT RAISED , LOW CONVEX , CONVEX , OR UMBONATE
EDGE: CRENATED, ENTIRE , LOBATE , UNDULATE , CILATE
OPACITY : TRANSLUCENT, TRANSPARENT OR OpAQUE
PIGMENT
HEMOLYSIS
CONSISTANCY: MUCOID , FRIABLE , firm , butyrous
SPECIMEN
DIRECT
MICROSCOPY
CULTURE
37O FOR 48 HRS

5. Morphology of bacterial colony

6. OVERVIEW STEPS OF CONVENTIAL
CULTURE METHODS:
• 1) sample collection
• 2) direct stain of sample
• 3) culture of sample on media for (24-48hrs)
• 4) stain of colony
• 5) biochemical of colony

7. BIOCHEMICAL REACTIONS
• Based on: colony morphology (gnb or gpc)
• 1) catalase : all organisms
• 2) GNB:
• A) Sugar fermentation
• b) indole test
• c)Methyl red test
• d) Voges proskauer test
• e)Oxidative fermantative test
• f)Phenyl pyruvic acid test
• g) citrate utilization test
• h) Urease hydrolisis test
• i) triple sugar iron test

8. • 3) Gpc :
• A) catalase: staphylococcus aureus
B)Dnase test: staphylococcus aureus
• C) CAMP( Christie-Atkins munch petersen): group B Streptococcos
• D) Bile eschuline test : Enterococcus
• E) Sugar fermentation test
inuline fermentation: pneumococcus
F) PYR test : Streptococcus pyogens and enterococcus
G) Bile solubility test : pneumococcus
H) Optochain test : pneumococcus
I) Bacitracin test : G-A (S) and G-B resistsnce

9. • Catalase : CATALASE
H2O2 H2O+O2
• catalase + : staphylococcus
• Catalase - : streptococcus

10. • Oxidase :
•
oxidase
• + organism: pseudomona
, vibrio, neisseria ,, bacillus etc
-ve organisms: enterobactericeae, acitenobacter etc

11. • Indole test :tryptaphanase enzyme breaks
aminoacid tryptophane
•
organism + : E.coli , vibrio clolerae
- Organism : klebsiella pneumoniae ,pseudomonas , salmonella
37oc for
48 hrs
Kovac’s (para
dimethylamino
benzaldehyde)

12. • Citrate :
• Utilized citrate as sole source of
carbon
• Butt and slant
• Media :
• Simmon’s citrate agar : colour change
to blue due to bromothymol blue
• Koser’s medium : turbit
• + organisms : klebsiella pneumoniae ,
citrobacter , enterobacter
• - organisms: E.coli , shigella

13. • Urea :
• Urease urea ammonia alkaline
• Media : Christensen’s urea media (phenyl red)
• + organisms:proteus , helocobacter pylori
• - organisms: E.coli , shigella

14. • Triple sugar iron test : gram negative bacteria
• Comosition
• 3 sugar : glucose , sucrose and lactose
• Indicator : phenyl red
• Ferric chloride : H2S (Hydrogen sulfide)

15. • Interpretation :
• K = red (alkaline)
• A= yellow (acidic)
• K/A= glucose only
• A/A=glucose , lactose/sucrose
• K/K= non formontive
•
k/k
A/A K/A
K/A
H2S
K/A
H2S

16. • Methyl red test :
sufficient acid during fermentation of glucose at ph4.5
+ organism : E.coli , listeria monocytogenes
-organism :klebsiella , enterobacter
37oc for
48 hrs
Methyl red
Glucose phosphate broth

17. • Glucose phosphate broth
37oc for
48 hrs
1ml 40% KOH &
3ml 5% alpha
nepthonal
acetylmethyl carbinol with glucose acetylmethyl carbinol converted
to diacetyl after adding of ∝- naphthol, strong alkali (40% KOH)
The diacetyl containing compounds found in the peptones of the broth then condense
to form a pinkish red polymer
Voges–Proskauer (VP) Test

18. • Nitrate reduction test:
• Nitrate reductase
• Nitrate Nitrite
•
37OC FOR 24
HRS
6-8 drops of nitrite reagent A
6-8 drops of nitrite reagent B
NITRATE POSITIVE : ALL ENTEROBACTERIACEAE
NITRATE NEGATIVE: HAEMOPHILUS DUCREYI
NITRATE BROTH

19. • Phenyl pyruvic acid test (PPA):
• phenylalanine deaminase
• PHENYLALANINE PYRUVIC ACID (10% ferric chloride)

20. Automated culture techniques
• Purpose
• A) culture
• B) identification
• C) AST

21. 1) AUTOMATED BLOOD CULURE
TECHNIQUES
Incubate
Microbial growth
indicated
By machine
More sensitive
Rapid
less labour

22. • tryptic soy broth
• brain hear infusion broth
polymetric beads : nutrilized antibiotics in blood
sensor
microorganism co2 Hydrogen
INCREASES (H) DECREASE PH
react

23. AUTOMATED STSTEM FOR BACTERIAL
IDNTIFICATION
• MALDI-TOF : (MARIX ASSISTED LASER
DESORPTION/IONIZATION TIME OF FLIGNT)
• VITEK -2: automated identification and AST
system