Obtaining Pure Microbial Culture

1. Streak or spread plate
2. Dilution plating
3. Dilution culturing

Streak plate or spread plate

This will be discussed under the section “practicals”.

Dilution plating

A series of test tubes containing 9 ml of sterile distilled water or 0.85% NaCl, peptone water and Ringer's solution has to be prepared which are called dilution blanks.1ml of the initial or original sample is transferred into the 1st test tube (10-1).Shake.Next 1ml of 10-1 solution is transferred into the next test tube (10-2). This procedure is carried out for another 5/6 test tubes. A sterile pipette is used to transfer solutions at each step. Then a pour plate is made for each and every dilution. (will be discussed at practicals section).

Dilution plating

Culturing Microorganisms

There are various culture media. Mixed culture, pure culture and in vitro ( cultured in containers), in vivo (grown in live animal and plant tissue).Culture medium basically requires a carbon source, nitrogen source, macronutrients, micronutrients, growth factors, gelling agents, antibiotics and dyes. There are two gelling agents agar and gelatin.

Agar is made out of red algae which have a melting point of 85 Celsius and a complex mixture of polysaccharides extracted from red algae. It is insoluble in cold water and sets between 32-39 Celsius. Gelatin is made out of hooves of animals. Thus high in protein. Melting point 30-35 celsius and readily digests by some bacteria.

Antibiotics are generally added before pouring into plates to inhibit a specific group of microorganisms. Since streptomycin is heat resistant it is added before heating whereas penicillin is heat sensitive thus added after heating.

Natural media

First used by Leeuwenhoek. This medium is prepared using peppercorns in water, hay in water and also meat in water. These are called infusions. Since the composition is not known it is difficult to reproduce.Eg; PDA, NA, Malt extract.

Artificial media

First used by pasture. Can be reproduced.Eg; Capex-dox nutrient.

• NaNO3  2g
• K2HPO4  1g
• MgSO4.7H2O  0.5g
• KCl  0.5g
• FeSO4  0.01g
• Sucrose 30g
• Agar 20g
• Distilled water 1L  

Basal media

Both natural and synthetic media.This suite for broad ranges of nutritionally undemanding groups of microorganisms.Eg; Cook’s no 2.

Selective media

A media that favor the growth of one type of specific microorganism while inhibiting the growth of undesirable microorganisms. By adding or removing of certain nutritional substances can separate one organism from another.

Eg; Coliform bacteria, Salmonella typhi.

Differential media

A culture media that distinguish different types of microorganisms by their metabolism of media components and colony appearance.Eg; Eosin Methylene blue agar, Yeast mannitol agar.

Suppressive media

Suppression of bacteria and encouragement of fungal growth.

Eg; Martin’s medium. This medium contains the antibiotic streptomycin and the dye rose Bengal. Rose Bengal suppresses bacterial growth as a result fungi grow.

Gram Staining Procedure

Gram stain is a method of differential staining. Gram stain was discovered and introduced by Christian Gram in 1884. This divides bacteria into two groups, gram positive and gram negative, depending on the presence of peptidoglycan in their cell wall. Bacteria lacking peptidoglycan termed as gram negatives and gram positives are the bacteria who contain peptidoglycan. Peptidoglycan can absorb dyes hence the presence of peptidoglycan gives color. 

Gram staining method has two particular dyes. Primary dye is the ammonium oxalate crystal violet which stains all cells with purple. Mordant is a substance that strengthens the union between the dye and the substrate or fixes coloring material. Gram’s iodine solution used as the mordant. Decolorizing agent 95% alcohol removes the primary stain from gram-negative cells and gram positives retain the primary dye. Safranin a secondary dye is used to stain the colorless gram-negative cells. Hence gram-positive cells appear in purple whereas gram-negative cells show a color of pink to red. This differential behavior has been explained by; the cell wall composition of gram negatives and positives and differential permeability to alcohol insoluble dye, iodine complex.

Gram Reaction 

This method is to distinguish gram negatives and gram-positive bacteria. This comparison depends on the presence of peptidoglycan in the cell wall.

Staining Techniques to Observe Stained Cells

Microorganisms are invisible to the naked eye. Therefore for the purpose of studying, they are cultured. Staining will kill microorganisms. Dyes are used to staining and smears are prepared. Most commercial dyes are salts in which one of the ions are colored. Eg: Methylene blue (MBCl).

MBCl   =   MB⁺ + Cl^-

There are two kinds of dyes. Basic dyes and acidic dyes. Methylene blue, crystal violet, carbon fuchsin are basic dyes in which the positive ion gives the color. Basic dyes will stain the acidic components of the cell such as the nucleus.

Salts in which the negative ion gives the color are called acidic dyes, stains basic components of the cell such as cytoplasm and granules. Eg:  Safranin, Congored, and Acid Fuchsin.

Methods of Staining

• Simple staining

A single dye application.Mainly basic dyes such as Methylene blue, crystal violet, carbolfuchsin used.

• Differential staining

Use more than one dye. This method distinguishes one type of cell from another.

Eg: Gram stain, KOH procedure (gram reaction).

Negative Staining and Hanging Drop Procedure

Observation of living cells follows below procedures.

Negative Staining

Microorganism will not stain in this method, but the medium/environment will stain. Staining will kill the microbe hence negative staining provides us the facility to observe the live cells. This method will be discussed more under the topic practical.

 Medium is stained but not cells

Hanging Drop Procedure

In this technique, a drop of medium containing cells is allowed to hang in the cavity slide. This method is used to study the motility of microorganism. There are two movements that can be observed, true movement and Brownian movement. Microorganism changes its position in a true movement whereas in Brownian movement the organism oscillates at its place and does not change the position in the field. Brownian movement can be explained based on the bombardment of small particles suspended in the fluid, with the cell. This method will be discussed more under the topic practical.