Exercise 6: Isolation of

Bacteria from Environmental

Samples

(Dumaguit, 2019)
Microbial life forms are distributed
everywhere
Ecosphere
- Atmosphere (gas)
- Hydrosphere (water)
- Lithosphere (rock & soil)

(Dumaguit, 2019)
COMPUTATION:

½ Strength Nutrient Agar 𝑔

23 𝒈
𝐿
Half Strength Nutrient Agar = = 𝟏𝟐. 𝟓
2 𝑳
𝑔
13.8 𝒈
𝐿
Half Agar Agar = = 𝟔. 𝟗
2 𝑳

Ex. 500 mL is needed

12.5𝑔 𝑥
x = 6.25𝑔
1000 𝑚𝐿 500 𝑚𝐿

6.25𝑔 + 6.9𝑔
(Dumaguit, 2019)
Airborne bacteria

Leave the plate exposed to

air for 10 minutes

Incubate: 24 – 72 hours

(Dumaguit, 2019)
 Flame Sterilize the L-shaped rod
• Place denatured alcohol in a 600 ml beaker
• Dip the L-rod into the alcohol
• Pass the L-rod all over the flame
• Allow the flame to extinguish on its own
• Cool for 20 seconds

(Dumaguit, 2019)
Bacteria in the Soil

Incubate: 24 – 48 hours

(Dumaguit, 2019)
Bacteria in the Pond Water

Place 1 drop of pond water

Spread drop using L-rod

Incubate: 24 – 48 hours

(Dumaguit, 2019)
Microorganisms on the skin

Use sterile cotton swab

Incubate: 35 C ; 24 – 48 hrs.

(Dumaguit, 2019)
Microorganisms on inanimate objects

Use sterile cotton swab

Incubate: 35 C ; 24 – 48 hrs.

(Dumaguit, 2019)
Exercise 7: Staining of
Bacterial Cells

(Dumaguit, 2019)
Most bacterial cells exist as colorless cells in nature

(Dumaguit, 2019)
 Staining
Done to provide contrast between the bacterial
cells and the background

(Dumaguit, 2019)
 Stain
 A Salt (made up of ions opposing in charge)
 Na + ; Cl -

(Dumaguit, 2019)
Basic Stain – Can color cells
Chromophore – Positively charged ion; has color
Bacterial cell – negatively charged; due to cytoplasmic membrane/ phosphate

• Crystal Violet
• Safranin
• Methylene Blue

(Dumaguit, 2019)
Negative Stain – Color the background
• India ink
• Nigrosin ink
• Acid fuchsin

(Dumaguit, 2019)
PROCEDURE:
SMEAR PREPARATION
Label the slide and draw a circle Place 2 loopfuls
Degreased
at the center of distilled
slides using
E. coli
Simple Staining acetone – allow water water at the
Dumaguit, D. to adhere and spread well center of the
2EBIO - 3 on the side
circle

Heat fix - Pass the Air dry to form a Aseptically transfer

slide over an open translucent film the bacteria to the
Opaque – too much of the
flame 3-5 times bacteria was used drop of water

(Dumaguit, 2019)
 Simple Staining
Provides a fast and easy way to determine cell
shape, cell size, and cell groupings

(Dumaguit, 2019)
PROCEDURE:
SIMPLE STAINING
Flood the prepared the
bacterial cell with the ff.
basic dye for 1 minute Drain and rinse the slide
E. coli – Crystal Violet
B. subtilis – Safranin
M. luteus – Safranin

Blot dry using a paper

Air dry
towel

(Dumaguit, 2019)
 Simple Staining

Escherichia coli Bacillus subtilis Micrococcus luteus

Stain: Crystal Violet Stain: Safranin Stain: Safranin
Cell Shape: Bacillus Cell Shape: Bacillus Cell Shape: Coccus
Cell Arrangement: Clusters Cell Arrangement: Chain Cell Arrangement: Clusters
Negative Positive Positive
(Dumaguit, 2019)
 Gram Staining
Allows differentiation of bacterial species based
on their cell wall type and the color that they
acquire after the staining process
Gram positive (+) species – will retain the primary stain
Gram negative (-) species – accept the counter stain

(Dumaguit, 2019)
 Gram Staining
 Developed by Christian Gram in 1884
 Based on the difference among stained bacteria in
their resistance to the decolorization by an alcohol

(Dumaguit, 2019)
Gram Staining Stains

Crystal Violet – Primary Stain

a chemical that fixes a dye in or on
Gram iodine – Mordant a substance by combining with the
dye to form an insoluble compound

95% ethanol – Decolorizer a substance that removes color

Safranin – Counterstain

(Dumaguit, 2019)
GRAM POSITIVE
Teichoic Acids (-)

Lipopolysaccharides (-)

GRAM NEGATIVE

(Dumaguit, 2019)
PROCEDURE:
GRAM STAINING
Flood the prepared Decolorize with 95%
slide with Crystal Drain and Flood with iodine
(1 minute) ethanol
Violet rinse
(10–15 seconds max)
(1 minute)

Wash with H2O Drain and

Blot dry Flood with safranin
(tap water inside rinse
using a tissue (1 minute)
wash bottle)

(Dumaguit, 2019)
 Gram Staining

Escherichia coli Bacillus subtilis Micrococcus luteus

Stain: Crystal Violet Stain: Safranin Stain: Safranin
Cell Shape: Bacillus Cell Shape: Bacillus Cell Shape: Coccus
Cell Arrangement: Clusters Cell Arrangement: Chain Cell Arrangement: Clusters
Gram Reaction: Gram Negative Gram Reaction: Gram Positive Gram Reaction: Gram Positive
(Dumaguit, 2019)
 Endospore
 For survival
 Develops when cells are subjected to
environmental stress
 Rich with Calcium and Dipicolinic acid
 Resist staining, but once stain, strongly resist
decolorization and counterstaining

(Dumaguit, 2019)
 Endospore Staining
Schaeffer-Fulton method – employs steaming the
smear in malachite green as an intense stain
Vegetative Cells – Pink to Red
Endospores – Green

(Dumaguit, 2019)
Endospore Staining Stains
Malachite Green – Primary Stain
Safranin – Counterstain

(Dumaguit, 2019)
PROCEDURE:
ENDOSPORE STAINING
Saturate the
Steam while holding Drain the excess
bacterial smear
the slide above a small dye and cool for a
with malachite
flame for 5 minutes few minutes
green (Do not allow to boil or to dry)

Rinse and Counterstain with Rinse

(water – decolorizes the
blot dry safranin (1 minute) vegetative cell)

(Dumaguit, 2019)
 Endospore Staining

Vegetative Cell

Endospore

(Dumaguit, 2019)
Exercise 8: Identification of
Bacteria

(Dumaguit, 2019)
 Bacteria once isolated is
Characterized by:
1. Gram Stain Reaction
2. Cultural Characteristics
Identified Based on:
1. Various metabolic activities

Conventional methods – employ the use of tubes or miniaturized strips

containing the biochemicals
Automated methods – shorten the turn around time to 4-6 hours

(Dumaguit, 2019)
 Using MacConkey Agar Plate
Growth in MAC indicates a Gram Negative bacteria

(Dumaguit, 2019)
 MacConkey Agar (MAC)
Lactose Fermentation

Escherichia coli (-) Bacillus subtilis (+)

MacConkey Agar Pink surface with pink colonies No Growth
Lactose Fermenter with Bile Non Lactose Fermenter
Precipitation
BIOCHEMICAL TEST RESULT: POSITIVE (+) NEGATIVE (-)
(Dumaguit, 2019)
 Using Mannitol Salt Agar Plate
Growth in MSA indicates a Gram Positive bacteria

(Dumaguit, 2019)
 Mannitol Salt Agar (MSA)
Mannitol Fermentation

Staphylococcus aureus (+) Bacillus subtilis (+)

Mannitol Salt Agar Yellow surface around the colonies Yellow surface around the colonies
Mannitol Fermentation Mannitol Fermentation

BIOCHEMICAL TEST RESULT: POSITIVE (+) POSITIVE (+)

(Dumaguit, 2019)
 Starch Agar (SA)
Starch Hydrolysis

Bacillus subtilis Staphylococcus aureus

Starch Agar Clearing around the colony Black color around the colony
Hydrolyzed starch Did not hydrolyzed starch

BIOCHEMICAL TEST RESULT: POSITIVE (+) NEGATIVE (-)

(Dumaguit, 2019)
 Eosin Methylene Blue Agar
Lactose Fermentation and Green Metallic Sheen

E. Coli
Fermented Lactose (+)
With green metallic sheen (+)

B. subtilis Fermented Lactose (+)

No growth (-) Green metallic sheen (+)
No green metallic sheen (-)

E. coli

(Dumaguit, 2019)
 Tryptone Broth (TB)
Indole Production
Add 5 drops of Kovac’s Reagent then mix
gently by tapping the tube

Pink color in Kovac’s No pink color in

layer in less than 1 upper layer
minute
Did not produced
Produced indole indole

Escherichia coli Enterobacter aerogens

POSITIVE (+) BIOCHEMICAL TEST RESULT NEGATIVE (-)
(Dumaguit, 2019)
 Simmon Citrate’s Agar (SCA)
Citrate Utilization

Change in color Remained

from green to color green
blue Did not
Utilized Citrate utilized Citrate

Enterobacter aerogens Escherichia coli

POSITIVE (+) BIOCHEMICAL TEST RESULT NEGATIVE (-)

(Dumaguit, 2019)
 A – Acidic
A˚ – Acidic with gas production
K- Alkaline
NC – No change (Dumaguit, 2019)
 Triple Sugar Iron Agar(TSI)

Escherichia coli
Both slant and butt are Both slant and butt are Slant is pink and butt is Both slant and butt are
yellow with crack yellow yellow and covered with red - pink
blackening

A/A˚ K/A with H2S K/K

A/A
production (Dumaguit, 2019)
 Ornithine Decarboxylation
Is a result of the stimulation by the acid production from the
fermentation of dextrose

(Dumaguit, 2019)
 Motility Indole Ornithine (MIO)
Motility, Indole Production, Ornithine Decarboxylation Test
Add 5 drops of Kovac’s Reagent then
mix gently by tapping the tube

Growth extending Growth does not extend

from the stab line from the stab line
Has motility No motility

Purple medium Yellow medium

Can Decarboxylate Can’t Decarboxylate
Ornithine Ornithine

Red color at the top Red color at the top in 2

in 2 minutes minutes
Produces Indole Does not produce Indole
Escherichia coli Micrococcus Luteus
POSITIVE (+) BIOCHEMICAL TEST RESULT NEGATIVE (-)
(Dumaguit, 2019)
Nitrate Broth (NIB)
Nitrate Reduction
Add 5 drops of
Nitrate A and
Nitrate B

Pink to red color

No color change
change in 1 min.
Positive

Add Zinc Dust

Pink in less than 1

No change in color
minute
Negative Positive
(Dumaguit, 2019)
 Nitrate Broth (NIB)
Nitrate Reduction

Change in color Change in color

after addition of after addition of
Nitrate A and Nitrate A and
Nitrate B Nitrate B
Reduces nitrate Reduces nitrate

Escherichia coli Bacillus subtilis

POSITIVE (+) BIOCHEMICAL TEST RESULT POSITIVE (+)

(Dumaguit, 2019)
 Nutrient Gelatin (NG)
Gelatinase

Bacillus subtilis Enterobacter aerogens

Liquified gelatin Solid gelatin

Produces Does not produce
gelatinase gelatinase

POSITIVE (+) BIOCHEMICAL TEST RESULT NEGATIVE (-)

(Dumaguit, 2019)