microbiology basic

Preparation, Dispensing and Sterilizing of Media for Cultivation of Microorganism


GS/M.Sc./FOOD/3608/08

B.K.K.K.Jinadasa

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Introduction

Medium is defined as any substrate or material that will enable microorganisms to grow and multiply. A common nutrient medium used for culturing microorganisms are exist in three forms i.e. liquid, semisolid and solid medium. In liquid medium all nutrients are added to water. It is also called broth e.g. nutrient broth. The liquid medium can be made into solid or semisolid medium by adding different quantity of solidifying agents like agar-agar, gelatin, silica gel, etc.

Example: nutrient agar, potato dextrose agar, martine rose Bengal agar etc.

The medium should contain all the essential substances required by microorganisms to be cultured. It should provide carbon, nitrogen, water source in addition to minerals and other growth factors for the growth of microorganisms. The basal nutrient medium can be supplemented with different substances such as sugars, proteins, inorganic salts etc. to satisfy the requirement of a particular organism.

Besides providing different substances required by microorganisms, the pH of medium should be adjusted at the optimum pH of microorganism to be culture. If pH is not adjusted, the growth of the microorganisms may be inhibited. In solid medium, agar should be added only after adjusting the pH.

On the basis of chemical components used for preparation of media, it is classified into three categories.

1) Synthetic media: media in which all the constituents are chemically defined. They are generally used to study the specific nutritional requirements of different microbes.

2) Complex media: media in which different components and their composition are incompletely defined. E.g. beef extract used in nutrient medium is chemically complex.

3) Natural medium: substances of natural origin that favours microbial growth are used in natural media. E.g. milk

Media can also be classified on heir functional properties.

1) Simple media: it is a common media used for cultivation of most of the microorganisms. E.g. nutrient agar

2) Differential media: these types of media are used to distinguish microorganisms that differ in their specific property. E.g. EMB agar, starch agar, blood agar

3) Selective media: these types of media selectively allow the growth of particular type of organisms and prevent the growth of the most of other microbes. E.g. martine rose Bengal agar.

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4) Selective differential media: these type of media allow only a particular type of organisms to grow that can also be further distinguish based on their property. E.g. Mc.Conkey agar.

1.1. Preparation, dispensing and sterilization of PDA (Potato dextrose agar) and NA (Nutrient agar)

Potatoes – 50 g

Agar - 5 g

Sucrose - 5 g

Distilled water – 250 ml

Procedure

Potatoes were peeled and cut into small pieces. Out of that 100g of peeled potatoes were taken Distilled water was added to it and boiled for 20 minutes. Potato extract was filtered through a muslin cloth to a conical flask.

10g of agar was added to the conical flask heated and was shaken to dissolve.

10g of glucose was added and dissolved.

Then the contents of the conical flask were transferred to a 1litre measuring cylinder and volume was adjusted to 500ml using distilled water.

pH was adjusted to 5.6 using 1N HCl (drop wise)

Above solution was autoclaved at 121°C (15psi) for 15 minutes.

8 ml of tartaric acid was added into 500ml of PDA (dissolve 1g of tartaric acid in 10ml of distilled water and sterilized. 1.6 ml of that solution is added to 100ml of PDA)

PDA media was poured in to Petri plates at 55°C - 60°C, before the media gets solidified.

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1.2. Preparation, dispensing and sterilization of Nutrient agar

Nutrient Agar is a complex medium. It supports the growth of a wide range of microorganisms. Nutrient media contain all the elements that most bacteria need for growth and are non-selective. The amino acid source contains a variety of compounds with the exact composition being unknown.

Beef extract is the commercially prepared dehydrated form of autolysed beef and is supplied in the form of a paste.

Peptone is casein (milk protein) that has been digested with the enzyme pepsin. Peptone is dehydrated and supplied as a powder.

Peptone and Beef Extract contain a mixture of amino acids and peptides.

Ingredients

Marmite or beef extract – 0.3 g

Peptone –0.5 g

Agar- 1.9 g

Distilled water- 100 ml

Procedure

Ingredients were suspended in 100ml of distilled water and boiled to dissolve completely.

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Discussion

When preparing potato dextrose agar, have to add antibacterial agents to inhibit the growth of bacteria .Chlorumphenicol or tartaric acid can be used as an antibacterial agent.

Containers used for media must have vented tops and should be capable of holding at least 20% more than the intended volume of medium, to allow for expansion during sterilization. One litre capped bottles to be very convenient for preparing large quantities, to facilitate cooling, handling, and pouring. Surface area of the liquid should be large enough to prevent superheating.

Agar does not distribute uniformly when melted. A safe way to ensure a uniform distribution for pouring plates or tubes is to drop a magnetic stir bar in the flask or bottle, then gently stir the medium after sterilization, while it cools. Stirring distributes the agar evenly.

1.3.Staining of microorganisms

Introduction

Living Bacteria, like most other cells, are essentially transparent, and must be stained in order to be easily visualized under the microscope. The specimen must be first spread out on a clean slide, heated gently to fix it to the slide, and then stained with an appropriate dye. Bacteria tend to be negatively charged, therefore positively-charged or basic dyes will bind to and stain them.

Staining techniques are widely used to visualise components under the light microscope, for the differentiation and identification of microorganisms.

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Types of staining

Simple staining of microbial cell

When a single staining-reagent is used and all cells and their structures stain in the same manner, the procedure is called simple staining procedure. They are also referred as monochrome stains. This procedure is of two types: positive and negative. In positive staining, the stain (e.g., methylene blue) is basic (cationic) having positive charge and attaches to the surface of object that is negatively charged. In negative staining, the stain (e.g., India ink, nigrosin) is acidic (anionic) having negative charge and is repelled by the object that is negatively charged, and thus fills the spaces between the objects resulting in indirect staining of the object. Some of the most commonly used dyes are methylene blue, carbolfuchsin, and crystal violet. Simple stains allow one to distinguish the shape (morphology) of the bacteria.

Differential staining of microbial cell

When more than one staining reagents are used and specific objects (e.g., specific microorganisms and or particular structure of a microorganism) exhibit different staining reactions readily distinguishable, the procedure is called as differential staining. The most widely used differential staining in microbiology are Gram-staining and acid-fast staining.

The Gram staining method is named after the Danish bacteriologist Hans Christian Gram (1853 – 1938) who originally devised it in 1882 (but published in 1884), to discriminate between pneumococci and Klebsiellapneumoniae bacteria in lung tissue. It is differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. This reaction divides the eubacteria into two fundamental groups according to their stainability and is one of the basic foundations on which bacterial identification is built. Gram staining is not used to classify archaea, since these microorganisms give very variable responses.

a) Indirect staining or negative staining of microbial cell

These are the dyes which stain only the background, e.g., nigrosin or India ink used either for observing mucilaginous covering enveloping bacteria (capsules) or certain spores of fungi or cells of unicellular animals. Negative staining is a technique by which bacterial cells are not stained, but are made visible against dark background. Acidic dyes like eosin and nigrosin are employed for this method.

b) Selective Stains.

These stains are used for special purposes, to stain particular parts of the organism such as spores, metachromatin granules, flagella, nuclei, etc. This type of stain is used to identify particular cellular structures. For example, a flagellar stain enables us to see the flagellar filaments which some cells use for movement; a spore stain to detect spores; likewise, a

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capsular stain used to visualise capsule material (generally a polysaccharide coat that some cells produce).

Materials and Reagents required and the procedures

1.3.1. Simple staining

Materials and Reagents:

Cultures of required organisms, Clean glass slide, Inoculating loop and needle, Spirit lamp,

Staining tray, Metyhylene blue, Crystal violet, Carbolfuchsin

Procedure:

Microscopic slides were washed with detergents and dried. A small clean drop of water was placed on the slide with the help of an inoculating loop. This water drop was inoculated by a portion of a colony of the given culture using a sterilized inoculating loop. A thin smear was prepared by this drop and the needle was flamed. Slide with smear was dried by moving it over the flame for two or three times. Each fixed smear was covered with approximately five drops of one of the stains for a designated time as indicated below.

Loffler’s methylene blue : 1-2 min

Crystal violet : 30 – 60 sec

Carbolfuchsin : 15 – 30 sec

After keeping the above designated time, excess dye was removed by washing with a gentle stream of running tap water and blot-dried using a blotting paper. Stained preparations were observed under the oil immersion objective of the microscope and significant differences in cell shape and arrangements were observed.

1.3.2. Gram staining

Materials and Reagents:

Bacterial cultures, Inoculating loop, Glass slides, Crystal violet, Gram’s iodine, 95% ethanol

Safranin

Procedure:

Thin heat fixed smears of provided bacterial cultures were prepared. Smears were covered with

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crystal violet (basic dye) for 1 minute and then washed with water for a few seconds and drained off excess water. Smear was flooded with Gram’s iodine solution (mordant) for 1 minute. Washed with water and drained. Carefully decolorized with 95% alcohol (decolorizing agent) until no more stain comes away (about 30 seconds). Then the slide was washed with tap water, drained and covered with counter stain safranin solution for 30 seconds. Slide was washed gently with water, blot dried and observed under the oil immersion objective.

Gram Staining Technique

1.3.4. Results

Simple staining

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(a)Smear stained with Loffler’s methylene blue:

Blue colour, coccus shaped cells arranged as chains were observed under the microscope.

(b) Smear stained with Crystal violet:

Purple colour, coccus shaped cells arranged as chains were observed under the microscope.

(c)Smear stained with Carbolfuchsin:

Red colour, coccus shaped cells arranged as chains were observed under the microscope.

Gram staining

Streptococcus cells were observed as purple colour, coccus cells.

E. coli cells were observed as pink colour, short bacillus in shape.

(a) (b)

Bacterial appearance under Gram Staining (a) G +Cocci (b) G –Rods

1.3.5. Discussion and Conclusion

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The observed cell appearance was Bluish colouredCoccusand Pinkishcolour Rods (Bacillus). Generally Streptococcus cells are Gram positive and E. coli are Gram negative found in the environment.

Most staining procedures are performed on fixed cells. Fixing, or heating the dried cells on the slide, kills the bacteria and causes them to stick to the slide thereby prevent washing off during washing steps. This is resulted some artifact on the smear.

The smear preparation should very much concern and too thick layering of staining solution will not allow lights to pass through the object and dye will crack on drying. Also too thin film will not give good contrast. Bacteria take up the Gram stain differently because they differ in cell wall composition. Gram-positive bacteria have a thick cell wall layer. Alcohol does not readily penetrate to decolorize the cell wall of the previously applied crystal violet stain. Gram-negative cells have a thinner cell wall through which the alcohol readily penetrates. The crystal violet is removed from these cell walls that are then stained with the safranin counterstain.

1.4. Isolation of Pure Cultures of Bacteria and Fungi from the Environment.

1.4.1 Serial dilution and culture techniques

A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometricprogression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M, 0.01 M, 0.001 M... Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments resulting in concentration curve with a logarithmic scale. As stated earlier, this method is commonly used to obtain pure cultures of those microorganisms that have not yet been successfully cultivated on solid media and grow only in liquid media. A microorganism that predominates in a mixed culture can be isolated in pure form by a series of dilutions.

1.4.2. Procedure (pour plate culture)

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http://en.wikipedia.org/wiki/Geometric_progression


1ml from 10 -3 to 10 -5 dilutions were transferred separately into sterile Petri plates. (Three plates from each dilution) and 15 ml of sterilized nutrient agar medium is poured to each plate.

Plates were shaken gently in order to spread the sample.

Plates were incubated at 37°C for 48 hours.

1.4.3.Procedure (streak plate culture)

Loop full of 10 -3 to 10 -5 dilutions were taken and streaked on the solidified nutrient agar and PDA separately. (Three plates from each dilution).

Plates were incubated at 37°C for 48 hours.

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microbiology basic

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