Microbiology and Parasitology Premid

7/31/2019 Microbiology and Parasitology Premid

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Gram staining by Hans Christian Gram

is a method of differentiating bacterialspecies into two large groups (Gram-positiveand Gram-negative).

It is based on the chemical and physical

properties of their cell walls. Primarily, itdetects peptidoglycan, which is present in athick layer in Gram positive bacteria.

A Gram positive results in a purple/bluecolor while a Gram negative results in apink/red color.

The Gram staining method

1. A small sample

of a bacterial

culture isremoved from a

culture.

The bacterial

suspension is

smeared ontoa clean glass

slide.

The bacterial

smear is thendried slowly at

first and then,

when dry,

heated for a

few seconds

Once cool, the slide

is flooded with a

stain called Crystal

Violet . The stain is

left on the slide forabout 1 minute. This

stains all the

bacteria on the slide

The Crystal

Violet is gently

washed off theslide with

running water

The bacterial

smear is then

treated with

Gram's solutionwhich consists of 1

part iodine, 2 parts

potassium iodide,

and 300 parts

water.

After about 30 seconds

the slide is gently

rinsed with ethyl

alcohol which causesthe dye-iodine complex

to be washed out of

some bacteria but not

others. This is called

decolourisation.


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a colour which

contrasts with the

blue-black colour of

the Gram-positive

cells. The staincommon used is

safranin which is

red. This is called

counterstain.

The counter

stain is left on

the smear for

about 30-60seconds and

then gently

rinsed away

with running

water.

After the

counterstain has

been rinsed off, the

slide is placed

between some

absorbent paper

and the excess

water gently

blotted off.

a drop of immersion

oil is placed on the

stained bacterial

smear.

The slide is then

placed on a

microscope stage

focus under oil

immersion objective

Typical Gram-positive bacteria

staphylococci such as Staphylococcus

epidermidis and Staphylococcus aureus which

is a common cause of boils streptococci such as the many species of oral

streptococci, Streptococcus pyogenes which

causes many a sore throat and scarlet fever

and Streptococcus pneumoniae which causes

lobar pneumonia

clostridia such as Clostridium tetani

which cause tetanus (lockjaw)

actinomyces such asActinomycesodontolyticus which is found in mouths

species of the genus Bacillus such as

Bacillus subtilis which are common

microbes living in soil


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Typical Gram-negative bacteria

the bacilli that cause

whooping cough, Bordetella pertussis

typhoid, Salmonella typhi

cholera, Vibrio cholerae gut-dwelling Escherichia coli

Acid Fast Staining by Ziehl Neelsen

Acid-fastness is a physical property of certain

bacteria, specifically their resistance to

decolorization by acids during staining

procedures. The high mycolic acid content of certain

bacterial cell walls, like those of

Mycobacteria is responsible for the staining

pattern of poor absorption followed by high

retention.

Mycobacterium tuberculosis

d. Lipases attacks fats and break them downinto glycerol and fatty acids

e. Zymases changes sugars to alcoholf. Oxidases catalyse oxidation, ex. Alcohol to

acetic acid

g. Dehydrogenases catalyze anaerobicoxidations, which removes hydrogen

h. Coagulases produce coagulation in liquidprotein

i. Reductases changes hydrogen peroxide towater and molecular oxygen

2. BASED ON RESULTS OF MICROBIAL GROWTH

a. Acid production in

media containing milk or

carbohydrates certain

bacteria form Lactic acid,

Acetic acid, Butyric acid,

Formic acid, or Proprionic

acid acids

Yellow Acid fermentation

of sugar

Red Alkaline due to

deamination of protein

IMViC TEST

- used to distinguish between different enteric

bacteria (Family Enterobacteriaceae)

- E. coli and Klebsiella are lactose fermenters

- Salmonella and Shigella are lactose

nonfermenters


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. Indol production

Bacteria that contain the

enzyme tryptophanase

can hydrolyze

tryptophan to indole,

pyruvic acid andammonia, can be

detected by adding

Kovacs reagent

- production of bright red

compound on the

surface of the medium

Methyl Red Test

Based upon the pH

concentration upon the

addition of Methyl red

Orange red color is (+) Yellow is (-)

Distinguishes:

Aerobacter aerogenes (-)

Escherichia coli (+)

Voges-Proskauer test

Based upon the

production of

acetylmethylcarbinol from

dextrose .

Useful to distinguish

Aerobacter aerogenes (+)

from Escherichia coli(-)

Citrate test

Determines ability of

bacteria to use citrate as a

sole carbon source for energyneeds

Bromothymol blue is used as

indicator

Escherichia coli (+)

b. Gas production

Observed in media

containing

carbohydrate such as

lactose or dextrose,

the gases formed are

carbon dioxide,

hydrogen, nitrogen,

hydrogen sulfide,

ammonia and

methane

c. Urease test

Bacteria containing

enzyme urease uses

nitrogen and carbon in

amide compounds such as

Urea.

Distinguish:

Proteus (+ ) from other

Nonlactose-fermenting

enteric bacteria like

Salmonella and Shigella (-)

d. Proteolysis

Liquefies gelatin,

coagulate serumEx. Proteus

Pseudomonas

d. Alcohol production

Produced by yeast,

molds and a few

bacteria


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e. Pigment Production

Carotenoids

produces yellow, red,

orange pigments

Ex. Sarcina,

Micrococcus Anthocyanins

produces red, blue and

intermediate shades

Ex. Actinomyces

Melanins

produces black,

brown

Ex. Azobacter,

Actinomyces

SMEAR PREPARATION AND STAINING

A BACTERIAL SMEAR IS

A DRIED PREPARATION

OF BACTERIAL CELLS ON

A GLASS SLIDE.

A SMALL AMOUNT OF

BACTERIAL GROWTH IS

TRANSFERRED TO A

DROP OF WATER ON A

GLASS SLIDE AND

MIXED, THE MIXTURE IS

SPREAD OUT EVENLY

OVER THE LARGE AREA

ON THE SLIDE

1. HANGING DROP

SLIDE AND BACTERIAL

MOTILITY

- Bacteria that possessflagella exhibit

flagellar motion

- Helically shaped

spirochete moves in a

corkscrew and

bending-type motion

- Gliding motion- slides

over moist surface

2. NEGATIVE STAINING

- uses India ink or

Nigrosin that will

stain around the

bacteria to produce adark background

3. GRAM STAINING

Is a method of

differentiating bacterial

species into two large

groups (Gram-positive

and Gram-negative).

Uses crystal violet as

Primary stain and

Safranin as counterstain

4. ACID-FAST STAINING

Ziehl-Neelsen techniqueemploys heat to drivecarbol fuchsin into the

cell, once stained theyare not easlydecolorized. The acidfastness is due tomycolic acid

Kinyountechniqueemploys a wettingagent (Tergitol 7)

5. ENDOSPORE

STAINING

SCHAEFFER-FULTON

uses malachite green

as primary stain and

safranin as counter

stain


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6. CAPSULE STAINING

- Anthonys method

employs crystal

violet as primary

stain and Coppersulfate as

decolorizer

PREPARATION OF CULTURE MEDIA

CULTURE MEDIUM refers to any material in

which microorganisms find nourishment.

- When the microorganisms in a culture are all of

the same species, it is called pure culture.- When two or more organisms are present it is

called mixed culture .

FORMS OF CULTURE MEDIA

- SYNTHETIC MEDIA

- NONSYNTHETIC MEDIA- DEHYDRATED MEDIA

May contain meat extract and agar

For tubes = 6 - 7 ml are necessary

For Plates = 10 ml are necessary

TYPES OF CULTURE MEDIA

Classification/Types Of Culture Media.

1. According to physical state.

a) Liquid media: fluid in nature, usuallyplaced in test tubes, for

example, nutrient broth.

b) Solid Media: Prepared by addingsolidifying agents like gelatin and agar tothe liquid medium, forexample, nutrient agar.

2. According To Composition

a)Simple Media: it contains only basicsubstance such as nitrogen , carbon andminerals that are essential for bacterialgrowth, for example, nutrient broth,

nutrient agar, peptone water .

b) Enriched Media: Some nutritionallyenriched material like blood, serum orasctic fluid is added to the medium,repuired for proper growth of somebacteria, for example, blood agar,chocolate agar.

c) Differential Media) it differntiate betweentwo groups of bacteria, for example, blood

agar, MacConkey's Medium

d) Selective Media: In this media an inhibitory

substance is added to the media whichprevents growth of all organisms except theone for which it is designed. for example,Lowenstein Jensen's medium.

e) Media used for biochemical reaction: Thismedia is used to detect different biochemicalreactions produced by different organisms. forexample, simmon citrate medium .


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Important Culture Media:

a) Nutrient agar

b) Blood agar

c)Chocolate agar

d)McConkey's Medium

e) Lowenstein jensen (LJ) mediumf) Loeffler's Coagulated Medium

g )Nutrient Broth

h) Mueller Hinton

i) Brain Heart Infusion

METHODS OF OBTAINING PURE CULTURES

1. POUR PLATE

- A series of dilution of bacterial culture in a

medium is made and then pouring in Petri

dish2. STREAK PLATE

- Melted agar is poured into Petri dishes and

allowed to harden

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Microbiology and Parasitology Premid