This document outlines the steps for identifying unknown bacterial samples, focusing on classification and identification processes. It details methods including morphological analysis, cultural characteristics, and biochemical testing, referencing Bergey's Manual as a key resource. The document provides specific techniques for evaluating characteristics such as shape, growth patterns, and metabolic reactions.

1. STEPS TO IDENTIFY UNKNOWN
BACTERIAL SAMPLES (PART-1)
Dr C R Meera
Assistant Professor & HOD
Department of Microbiology
St. Mary’s College Thrissur

2. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR
Classification Vs Identification ?
• Classification is the separation of organisms into
distinct groups
• Identification is the determination of correct
place of an organism in a previously established
plan of classification

3. • Bergey’s Manual has been the internationally
accepted reference for the identification of
bacteria since 1923.
• Bergey’s Manual of Systemic Bacteriology
arranges related bacteria into 33 groups called
sections
• Sections - Morphology, staining reactions,
nutrition, cultural characteristics, physiology,
cellular chemistry and biochemical test
results for specific metabolic end products.
Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR

4. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR
Steps involved in the Identification of bacteria
• Morphological
• Cultural characteristics
• Biochemical tests

5. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR
1. Morphological
Shape : Spheres, short rods, long rods,
commas, spirals, filaments,
Arrangement : Single, pairs, chains, clusters
Capsules : Present or absent
Gram stain : Positive or negative
Spore stain : Non spore or spore former
(Central, Sub terminal or Terminal spore)
Motility : Motile or non motile
Courtesy:microbeonline.com

6. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR
2. Cultural characteristics
On Nutrient agar plate
Colonies : Golden, yellow, white,
glistening
Growth : Absent (0), slight (+),
moderate (2+), abundant (3+)
Form : Circular, irregular, rhizoid
Margins : Entire, lobate, undulate, serrate,
filamentous
Elevation : Flat, raised, convex, umbonate
Density : Opaque, translucent

7. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR
2. Cultural characteristics
In Nutrient broth media
Surface growth: Ring, pellicle, none
Turbidity : Heavy, slight, none
Sediment : Abundant, scanty, none, granular, flocculent

8. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR
2. Cultural characteristics
On Special media
a) Differential / Selective media
Egs:
Mc Conkey agar :Inhibit G +ve bacteria; differentiate lactose
fermenters and non lactose fermenters
Mannitol salt agar: Promote halopiles like Staphylococcus,
inhibit the non halophiles
Eosin Methylene Blue (EMB) agar :Inhibit G +ve bacteria;
differentiate enteric lactose fermenters and non
fermenters as well as identify colon bacillus, E. coli
b) Enriched media
Eg:
Blood agar :Differentiate hemolytic microorganisms like
Streptococcus spp. from non hemolytic species
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9. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR
3. Biochemical Tests
a) Carbohydrate fermentation test: Red to yellow; with or without gas
b) IMViC tests
Indole production test : Formation of red layer
Methyl-red (MR) test : Presence of red color
Voges - Proskauer (VP) test : Deep rose (pink) color
Citrate utilization test : Color change of media from green to blue
c) Urease test : Red to deep pink
d) Nitrate reduction test : Red color
e) Catalase activity : Bubbles on the release of free oxygen gas (O2)
f) Oxidase test : Development of pink, then maroon and finally
black coloration on the bacterial colonies
g) Triple Sugar Iron (TSI) test : Alkaline (red) or/ acidic (yellow) color change
in slant and butt region; with or without gas
production

10. Steps to identify unknown bacterial samples; Dr C R Meera, St.Mary’s College, TCR