Gram staining by Hans Christian Gram

is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative). It is based on the chemical and physical properties of their cell walls. Primarily, it detects peptidoglycan, which is present in a thick layer in Gram positive bacteria. A Gram positive results in a purple/blue color while a Gram negative results in a pink/red color.

Once cool, the slide is flooded with a stain called Crystal Violet . The stain is left on the slide for about 1 minute. This stains all the bacteria on the slide

The Gram staining method

1.

A small sample of a bacterial culture is removed from a culture.

The Crystal Violet is gently washed off the slide with running water

The bacterial suspension is smeared onto a clean glass slide.

The bacterial smear is then treated with Gram's solution which consists of 1 part iodine, 2 parts potassium iodide, and 300 parts water.

The bacterial smear is then dried slowly at first and then, when dry, heated for a few seconds

After about 30 seconds the slide is gently rinsed with ethyl alcohol which causes the dye-iodine complex to be washed out of some bacteria but not others. This is called decolourisation.

 a colour which contrasts with the blue-black colour of the Gram-positive cells. The stain common used is safranin which is red. This is called counterstain.

The counter stain is left on the smear for about 30-60 seconds 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.

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

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

clostridia such as Clostridium tetani

which cause tetanus (lockjaw)

actinomyces such as Actinomyces

odontolyticus which is found in mouths

species of the genus Bacillus such as

Bacillus subtilis which are common microbes living in soil

Typical Gram-negative bacteria

the bacilli that cause

whooping cough, Bordetella pertussis typhoid, Salmonella typhi cholera, Vibrio cholerae gut-dwelling Escherichia coli

d. Lipases attacks fats and break them down into glycerol and fatty acids e. Zymases changes sugars to alcohol f. Oxidases catalyse oxidation, ex. Alcohol to acetic acid g. Dehydrogenases catalyze anaerobic oxidations, which removes hydrogen h. Coagulases produce coagulation in liquid protein i. Reductases changes hydrogen peroxide to water and molecular oxygen

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.

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

Mycobacterium tuberculosis

. Indol production
Bacteria that contain the enzyme tryptophanase can hydrolyze tryptophan to indole, pyruvic acid and ammonia, 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 (+)

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 (-)

Voges-Proskauer test
Based upon the production of acetylmethylcarbinol from dextrose . Useful to distinguish Aerobacter aerogenes (+) from Escherichia coli(-)

d. Proteolysis Liquefies gelatin, coagulate serum Ex. Proteus Pseudomonas

Citrate test Determines ability of bacteria to use citrate as a sole carbon source for energy needs Bromothymol blue is used as indicator Escherichia coli (+)

d. Alcohol production Produced by yeast, molds and a few bacteria

e. Pigment Production
Carotenoids produces yellow, red, orange pigments Ex. Sarcina, Micrococcus Anthocyanins produces red, blue and intermediate shades Ex. Actinomyces

2. NEGATIVE STAINING - uses India ink or Nigrosin that will stain around the bacteria to produce a dark background

Melanins produces black, brown Ex. Azobacter, Actinomyces

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

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

4. ACID-FAST STAINING Ziehl-Neelsen technique employs heat to drive carbol fuchsin into the cell, once stained they are not easly decolorized. The acid fastness is due to mycolic acid Kinyoun technique employs a wetting agent (Tergitol 7)

1. HANGING DROP SLIDE AND BACTERIAL MOTILITY - Bacteria that possess flagella exhibit flagellar motion - Helically shaped spirochete moves in a corkscrew and bending-type motion - Gliding motion- slides over moist surface

5. ENDOSPORE STAINING SCHAEFFER-FULTON uses malachite green as primary stain and safranin as counter stain

6. CAPSULE STAINING
- Anthonys method

Classification/Types Of Culture Media.

1. According to physical state.

employs crystal violet as primary stain and Copper sulfate as decolorizer

a) Liquid media: fluid in nature, usually placed in test tubes, for example, nutrient broth. b) Solid Media: Prepared by adding solidifying agents like gelatin and agar to the liquid medium, for example, nutrient agar.
2. According To Composition

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 .

a)Simple Media: it contains only basic substance such as nitrogen , carbon and minerals that are essential for bacterial growth, for example, nutrient broth, nutrient agar, peptone water . b) Enriched Media: Some nutritionally enriched material like blood, serum or asctic fluid is added to the medium, repuired for proper growth of some bacteria, for example, blood agar, chocolate agar.
c) Differential Media) it differntiate between

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

two groups of bacteria, for example, blood agar, MacConkey's Medium

d) Selective Media: In this media an inhibitory substance is added to the media which prevents growth of all organisms except the one for which it is designed. for example, Lowenstein Jensen's medium. e) Media used for biochemical reaction: This media is used to detect different biochemical reactions produced by different organisms. for example, simmon citrate medium .

TYPES OF CULTURE MEDIA

Important Culture Media:

a) Nutrient agar b) Blood agar c)Chocolate agar d)McConkey's Medium e) Lowenstein jensen (LJ) medium f) 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 dish 2. STREAK PLATE - Melted agar is poured into Petri dishes and allowed to harden