Media Preparation

Preparation of Nutrient Agar Plate

Aim

To prepare nutrient agar plates for cultivation of bacteria.

Principle

Nutrient Agar is a basal solid medium which support the growth of a variety of common heterotrophic bacteria. It is one of the general purpose media. It is used for the isolation of microorganisms and to do some sensitivity tests. The pH of the medium is 6.8-7.2. The neutral range of pH range aids the growth of a wide variety of microorganisms. The nutrient agar media consists of:

Peptones- They are protein hydrolysate prepared by partial proteolytic digestion of meat, casein, soya meal, gelatin and other protein sources. They serve as source of carbon, energy and nitrogen.

Beef Extract- They are prepared by extracting water soluble components of beef tissues. It is a complex mixture of proteins, carbohydrates, lipids and other biochemical constituents.

Yeast Extract- An aqueous extract of yeast cells containing vitamins and other growth factors.

Agar agar- A complex polysaccharide extracted from the algae Gellidium. It is a solidifying agent with gelling temperature of 45^oC and melting temperature of 95^oC. It is not a nutrient source and is resistant to enzymatic dehydration.

Materials Required

Beef extract, Yeast extract, peptone, sodium chloride, agar agar, distilled water, conical flask, weighing machine, measuring cylinder, heater.

Procedure

1. Beef extract, yeast extract, peptone and sodium chloride were weighed to the required amount and added to a known volume of water.

2. They were mixed well and dissolved.

3. pH was adjusted to 6.8-7.2.

4. The agar was weighed and added to the medium and dissolved by boiling.

5. After the agar was dissolved properly, the medium was poured to conical flask or tubes in the required amount.

6. These were plugged with cotton and autoclaved at 121^oC at 15 lbs pressure for 20 minutes.

7. The medium was then cooled to hand bearable temperature and poured into sterile petri plates.

Result

A nutrient agar plate was prepared.

 

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Preparation of Nutrient Broth

Aim

To prepare nutrient broth for cultivation of bacteria.

Materials Required

Beef extract, Yeast extract, peptone, sodium chloride, distilled water, conical flask, weighing machine, measuring cylinder, heater.

Procedure

1. Beef extract, yeast extract, peptone and sodium chloride were weighed to the required amount and added to a known volume of water.

2. They were mixed well and dissolved.

3. pH was adjusted to 6.8-7.2.

4. The medium was poured to conical flask or tubes in the required amount.

5. Those were plugged with cotton and autoclaved at 121^oC at 15 lbs pressure for 20 minutes.

6. The medium was then cooled to hand bearable temperature and poured into sterile test tubes as per requirement.

Result

A nutrient broth medium was prepared.

 

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Preparation of Agar Slant and Agar Deep culture tubes

Aim

To prepare nutrient agar slant and deep culture tubes

Principle

Autoclaved agar medium poured into culture tubes and placed in slanting position results in slant after solidification. If the culture tubes are kept erect on solidification, it gives deep tubes.

Materials Required

Nutrient agar medium, sterilised test tubes, test tube stand, cotton plug, Bunsen burner

Procedure

1. 8-10 ml nutrient agar medium is poured into each test tube and cotton plugs put.

7. Tubes were transferred to test tube stand and autoclaved at 121^oC at 15 lbs pressure for 20 minutes.

2. One set of test tubes were kept in slanting position and second set of test tubes kept in test tube stand.

3. Medium was allowed to solidify.

Result

Agar slant and agar deep culture tubes were prepared.

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Cultural Characteristics

Aim

To determine the cultural characteristics of microorganisms

Principle

Cultural characteristics describe various features of growth of bacteria on medium. When grown on a variety of media, microorganisms will exhibit differences in the macroscopic appearances of their growth. These differences, called the cultural characteristics, form the basis for their preliminary identification and classification into various taxonomic groups. These are determined by culturing microorganisms into different media like, nutrient broth, nutrient agar tubes, nutrient agar plates and nutrient gelatin.

1)      Nutrient Broth

The growth of microorganisms in broth can be described based on the distribution and the appearance,

a)      Amount – Scanty, moderate, abundant

b)     Turbidity - Uniform, finely dispersed growth throughout the medium

c)      Flocculent: Flaky aggregates distributed throughout

d)     Surface growth:  Ring, pellicle (pad like growth-thin, thick, smooth, granular)

e)      Sediment: Concentration of growth at the bottom of broth culture- granular, flaky or flocculent.

f)       Odour: Peculiar odour absent or present

 

2)      Nutrient Agar slants

The cultures grow along the line of inoculation on the surface of the slant after appropriate conditions of incubation. If a wavy line with growth along its length is observed, it means the culture has grown in the media. No line of growth shows the inability of the culture to grow in the media under the incubation conditions. The slant is observed for characteristics as follows:

a)      Abundance of Growth: The amount of growth is designated as none, slight, moderate or large.

b)     Pigmentation: Chromogenic microorganisms may produce intracellular pigments that are responsible for the coloration of the organism as seen in surface colonies. Other organisms produce extracellular soluble pigments that are excreted into the medium and also produce a color. Most organisms, however, are non-chromomeric and will appear white to gray.

c)      Optical Characteristics: Optical characteristics may be evaluated on the basis of the amount of light transmitted through the growth. These characteristics are described as: opaque - No light transmission, translucent - partial light transmission, transparent - full light transmission.

d)      Form: The appearance of the single line streak of growth on the agar surface is designated as follows:

(i) Filiform: Continuous, threadlike growth with smooth edges.

(ii) Echinulate: Continuous, threadlike growth with irregular edges.

(iii) Beaded: Non-confluent to semi-confluent colonies.

(iv) Effuse: Thin, spreading growth.

(v) Arborescent: Tree-like growth.

(vi) Rhizoid: Root-like growth.

3)      Nutrient Agar plates

These demonstrate well isolated colonies and are evaluated in the following manner:

a)      Size: Pinpoint, small, moderate or large

b)     Pigmentation: Colour of colony

c)      Form: The shape of the colony is described as,

(i)                 Circular – unbroken peripheral edge

(ii)              Irregular – indented peripheral edge

(iii)             Rhizoids – Root-like spreading growth

d)     Margin: The appearance of outer edge of colony is described as

(i)                 Entire – sharply defined, even

(ii)              Lobate – marked indentation

(iii)            Undulate – Wavy indentation

(iv)             Serrate – tooth like appearance

(v)               Filamentous – thread-like spreading edge

e)      Elevation: The degree onto which colony growth is raised on agar surface and is described as,

(i)     Flat- no elevation

(ii)  Raised – slightly elevated

(iii)                        Convex – dome shaped elevation

(iv) Umbonate – raised with elevated convex central region

f)       Opacity – Opaque, translucent, transparent

g)      Surface – Smooth, rough, papillate, ringed, fried egg, medusa head

 

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Preparation and Use of Differential, Selective and Enriched Media to Distinguish the Growth Characteristics of Microorganisms

Much of the study of microbes depends on its ability to grow in the laboratory and this is possible only if suitable culture media are available.  A growth medium or culture medium is a solid, liquid or semi-solid designed to support the growth of microorganisms.  Many different types of media are used in the microbiology laboratory.

Culture media may be classified into several categories depending on their composition or use. A chemically-defined (synthetic) medium is one in which the exact chemical composition is known. A complex (undefined) medium is one in which the exact chemical constitution of the medium is not known. Complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract.   Defined media are usually composed of pure biochemicals. A defined medium is a minimal medium if it provides only the exact nutrients (including any growth factors) needed by the organism for growth.

In general, these media can be divided into several categories, including nutritive, differential, and selective.  Nutritive media are defined as media types that support the growth of a wide range of microorganisms. Examples of nutritive media include nutrient agar.

Differential medium are those that distinguish microorganisms from one another based on growth characteristics when grown. Different organisms show visible differences in growth when placed on differential media. Examples include blood agar, Eosin Methylene Blue (EMB) agar, and MacConkey agar.

Selective medium types are formulated to support the growth of one group of organisms, but inhibit the growth of another. These media contain antimicrobials, dyes, or alcohol to inhibit the growth of unwanted organisms not targeted for study. Selective medium types include EMB agar, Mannitol Salt agar and MacConkey agar.

Some media may possess both selective and differential properties and can be powerful diagnostic tools in both medical and environmental settings.  

Enriched Media are supplemented with highly nutritious materials such as blood, yeast extract, and serum and is used for cultivating fastidious organisms. Examples are blood agar and chocolate agar.

Eosin Methylene Blue agar (EMB agar)

It contains the dyes eosin and methylene blue and inhibit Gram-positive organisms.  This medium is selective for Gram-negative species.  Lactose-fermenting organisms such as E. coli produce a black precipitate on EMB.  Their colonies will be either black or possess dark centers with transparent, colorless peripheries.  Non-lactose fermenters such as Proteus sp., Salmonella sp., or Shigella sp. appear pink or uncolored. 

MacConkey agar

This medium is also selective for Gram-negative species and differential with respect to lactose fermentation. MacConkey agar is used for the detection of coliforms and enteric pathogens based on their ability to ferment lactose.  Lactose-fermenting bacteria appear red to pink while non-lactose fermenting bacteria appear as colorless or transparent colonies.

Blood Agar

This is a nutritive medium with differential properties in respect to hemolysis. Blood agar permits demonstration of the hemolytic properties of certain microorganisms, such as streptococci, whose hemolytic activities are classified as Gamma Hemolysis, Alpha Hemolysis, and Beta Hemolysis  Complete breakdown of the RBCs is termed beta (β) hemolysis and is recognized by clearing around the colonies. Partial destruction of the RBCs leads to a greenish brown color on the agar and is termed alpha(α)  hemolysis. Gamma (γ) hemolysis is the term applied to growth on blood agar that causes no damage to the RBCs and no change in the medium.

Chocolate Agar

This is lysed blood agar. The name is itself derived from the fact that red blood cell (RBC) lysis gives the medium a chocolate-brown color. It is used for the isolation of fastidious organisms, such as Haemophilus influenzae.  The composition of Chocolate agar and Blood Agar is same and the only difference is while preparing Chocolate agar, the red blood cells are lysed. 

The lysis of RBC during the heating process releases intracellular coenzyme Nicotinamide adenine dinucleotide (NAD or V Factor) in to the agar for utilization by fastidious bacteria.  The most common species that require this enriched medium for growth are Neisseria gonorrhoeae, Neisseria meningitidis and Haemophilus spp.