Practical: NO 01 Preparation dispensing & sterilization of media for cultivation of micro organisms 1.

1 Introduction:A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens. There are different types of media for growing different types of cells.[ There are two major types of growth media: those used for cell culture, which use specific cell types derived from plants or animals, and microbiological culture, which are used for growing microorganisms, such as bacteria or yeast. The most common growth media for microorganisms are nutrient broths and agar plates; specialized media are sometimes required for microorganism and cell culture growth. Some organisms, termed fastidious organisms, require specialized environments due to complex nutritional requirements. Viruses, for example, are obligate intracellular parasites and require a growth medium containing living cells.

Nutrient media

a source of amino acids and nitrogen (e.g., beef, yeast extract)

This is an undefined medium because the amino acid source contains a variety of compounds with the exact composition being unknown. Nutrient media contain all the elements that most bacteria need for growth and are non-selective, so they are used for the general cultivation and maintenance of bacteria kept in laboratory culture collections. An undefined medium (also known as a basal or complex medium) is a medium that contains:
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a carbon source such as glucose for bacterial growth water various salts needed for bacterial growth

Defined media (also known as chemically defined media or synthetic media)
• •

all the chemicals used are known does not contain any yeast, animal or plant tissue.

Differential media
Differential media or indicator media distinguish one microorganism type from another growing on the same media.[5] This type of media uses the biochemical characteristics of a microorganism growing in the presence of specific nutrients or indicators (such as neutral red, phenol red, eosin y, or methylene blue) added to the medium to visibly indicate the defining characteristics of a microorganism. This type of media is used for the detection of microorganisms and by molecular biologists to detect recombinant strains of bacteria.


Minimal media
Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow "wild type" microorganisms. Minimal media can also be used to select for or against recombinants or exconjugants. Minimal medium typically contains:
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a carbon source for bacterial growth, which may be a sugar such as glucose, or a less energy-rich source like succinate various salts, which may vary among bacteria species and growing conditions; these generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the bacteria to synthesize protein and nucleic acid water

Supplementary minimal media are a type of minimal media that also contains a single selected agent, usually an amino acid or a sugar. This supplementation allows for the culturing of specific lines of auxotrophic recombinants.

Selective media
Selective media are used for the growth of only select microorganisms. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing.

Enriched media
Enriched media contain the nutrients required to support the growth of a wide variety of organisms, including some of the more fastidious ones. They are commonly used to harvest as many different types of microbes as are present in the specimen. Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40-45°C), which turns brown and gives the medium the color for which it is named 1.2 Potato Dextrose Agar (PDA) Potato dextrose agar is the most widely used medium for growing fungi and bacteria which attack living plants or decay dead plant matter. This is the commonest medium used to culture fungi. Specially this is a excellent medium for the detection of Fusaria sp. Because fungi needs, higher carbonic medium, pH slightly acidic (6.0-6.5) than bacteria


Ingredients Sucrose 10g; Potato 100g; Agar 10g; Distilled water 500ml Method  Potato was cleaned thoroughly peeled  Cut in to 1.5 cm cubs  500ml of water was added  Boiled for 1hr was stirred until all the agar got dissolved Observations

PDA Plate 1.3 Nutrient Agar Nutrient agar is a microbiological growth medium commonly used for the routine cultivation of non-fastidious bacteria. It is useful because it remains solid even at relatively high temperatures. Also, bacteria grown in nutrient agar grows on the surface, and is clearly visible as small colonies. In nutrient broth, the bacteria grow in the liquid, and are seen as a soupy substance, not as clearly distinguishable clumps. Nutrient agar typically contains. Ingredients Marmite or beef extract 3g; Peptone 5g; Agar 15-20g; Distilled water 1000 ml Method Suspended the ingredients in 1 liter of water and boiled to dissolve completely. Observation


Nutrient Agar Plate 1.4 Dispensing & sterilization of the media 15ml of the medium was poured in to each test tube or flask or bottle of capacity not more than 500ml while taking care not to make contact the medium with the mouth or the rim of the tube or bottle. Plug the mouth with non absorbent cotton wool & cover them with aluminum foils to prevent getting wet during sterilizing. Containers were then autoclaved for 15 minutes at 121 C. Sterilization of glass ware & apparatus Glassware could be sterilized either by dry method which requires keeping apparatus in an oven at 170-175 C for not less than 60 minutes or using wet method in an autoclave at 121 C for not less than 20 minutes. 1.5 Staining techniques Living bacteria are almost colorless & therefore they are not contrastable to become visible in water where they are suspended. The degree of the contrast between them & the surrounding medium can be greatly increased by staining them with dyes. Staining techniques are greatly used to visualize components under the light microscope for the differentiation & identification of microorganisms. Biochemical differences among microorganisms enable us to differentiate them by staining. Stains are dyes that have three characteristic functional groups. (1) An aromatic group (2) a chemical group called chromophore which lends a color to the aromatic group & (3) an


auxochrome group which allows the compound to get combined by either electro statically or covalently with the target thereby staining it. This process is typically irreversible. There are two types of dyes (A) Acidic dyes which have an affinity towards positively charged components in the cell as it has an anionic auxochrome group & (B) basic dyes which have an affinity towards negatively charged components in the cell as it has an cationic auxochrome group. Stain generally reacts with the microorganism only & stains the cell but not with the background. Certain stains can be used to color & identify the small cell components which would otherwise be unseen under the light microscope. 1.5.1 Simple staining A simple stain employs a single stain that is used primarily to examine shape & arrangement of cells. Basic stains such as Methylene blue & crystal violet are normally used for this type of staining. In simple staining bacterial smear is stained with a single reagent. Basic stains with appositively charged chromogen are preferred here as bacterial nucleic acids & cell wall components carry a negative charge that strongly attract & binds to the cationic chromogen. The purpose of simple staining is to elucidate the morphology & arrangement of bacterial cells. 1.5.2 Materials & reagents Cultures of required organisms, clean glass slides, inoculation loops & needles, spirit lamp, staining tray, Methylene blue, Crystal violet, Carbol fuchsin 1.5.3 Procedure Microscope slides were washed with detergents & dried. A loop full of water was placed on the slide with the help of an inoculation needle. Heat the needle until it gets red by flame & allowed it to reach to room temperature. A Petri dish was slightly opened & a portion of a colony was removed with the aid of the needle & it was emulsified in the drop of water on the slide with


circular motion of the needle. The water drop was then spread in to a thin layer & got heat fixed by moving it over a flame. Smear was covered with five drops of Carbol fuchsin & kept for 1520 seconds. Then excess dye was washed away with gently running water & blot dried using a blotting paper. Then the slide was examined under microscope.

Making a smear on a glass slide 1.5.4 Differential staining Deferential stain uses two contrasting dyes to distinguish between deferent types of organisms there are four terms generally used in multi dye staining. Those are primary stain, mordant, decolorizer & counter stain. 1.5.5 Gram staining The Gram staining method, named after the Danish bacteriologist who originally devised it in 1882 (published 1884), Hans Christian Gram, is one of the most important staining techniques in microbiology. It is almost always the first test performed for the identification of bacteria. The primary stain of the Gram's method is crystal violet. Crystal violet is sometimes substituted with methylene blue, which is equally effective. The microorganisms that retain the crystal violetiodine complex appear purple brown under microscopic examination. These microorganisms that are stained by the Gram's method are commonly classified as Gram-positive or Gram nonnegative. Others that are not stained by crystal violet are referred to as Gram negative, and appear red. 6

Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye during solvent treatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan and lower lipid content than gram-negative bacteria. Bacteria cell walls are stained by the crystal violet. Iodine is subsequently added as a mordant to form the crystal violetiodine complex so that the dye cannot be removed easily. This step is commonly referred to as fixing the dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of ethanol and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid layer enhances the leaching of the primary stain from the cells into the surrounding solvent. In contrast, the solvent dehydrates the thicker Gram-positive cell walls, closing the pores as the cell wall shrinks during dehydration. As a result, the diffusion of the violet-iodine complex is blocked, and the bacteria remain stained. The length of the decolorization is critical in differentiating the gram-positive bacteria from the gram-negative bacteria. A prolonged exposure to the decolorizing agent will remove all the stain from both types of bacteria. Some Grampositive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). Finally, a counter stain of basic fuchsin is applied to the smear to give decolorized gram-negative bacteria a pink color. Some laboratories use safranin as a counter stain instead. Basic fuchsin stains many Gram-negative bacteria more intensely than does safranin, making them easier to see. Some bacteria which are poorly stained by safranin, such as Haemophilus spp., Legionella spp., and some anaerobic bacteria, are readily stained by basic fuchsin, but not safranin. The polychromatic nature of the gram stain enables determination of the size and shape of both Gram-negative and Gram-positive bacteria. If desired, the slides can be permanently mounted and preserved for record keeping. 1.5.6 Materials & reagents used Bacterial cultures, Inoculation loops, glass slides, crystal violet, Gram’s Iodine, 95% ethanol, safranin. 1.5.7 Procedure.


A thin smear was prepared from bacterial cultures provided & fixed them using heat. Smear was then stained with crystal violet the primary stain & kept for 1 minute. Then it was washed with water & excess water was drained off. Then the smear was flooded with grams iodine solution which is used as the mordent & allowed to stand for 1 minute. Again the smear was washed with water & excess water was drained. It was then carefully decolorized with 95% ethanol for 30 seconds & was washed again with gently running water. Then the counter stain safranin was flooded over the smear & allowed to stand for 30 seconds. Then again was washed with running water, blot dried with blotting paper & observed under microscope. 1.5.8 Results & Observations

LEFT: Gram-positive bacteria staining violet or purple in the Gram staining procedure after treatment with Gram’s stains RIGHT: Gram Negative bacteria staining red or pink in the Gram staining procedure after treatment with Gram's stain. 1.5.9 Discussion The thickness of Gram positive cell wall and presence of more lipids in Gram negative cell walls have been more acceptable reasons for Gram stain reactions. It is believed that the positively charged crystal violet pass through the cell wall and cell membrane and binds to negatively charged components inside the cell. Addition of negatively charged iodine (in the mordant) binds to the positively charged dye and forms a large dye-iodine complex within the cell. Crystal violet interacts with aqueous KI-I2 via a simple anion exchange to produce a chemical precipitate. The small chloride anion is replaced by the bulkier iodide, and the complex thus formed becomes


insoluble in water. During decolorization, alcohol dissolves the lipid present in the outer membrane of Gram negative bacteria and it leaches the dye-iodine complex out of the cell. A thin layer of peptidoglycan does not offer much resistance either. The dye-iodine complexes are washed from the Gram negative cell along with the outer membrane. Hence Gram negative cells readily get decolorized. On the other hand Gram positive cells become dehydrated from the ethanol treatment, closing the pores as the cell wall shrinks during dehydration. The dye iodine complex gets trapped inside the thick peptidoglycan layer and does not get decolorized. Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-negative bacteria (Gram-variable). When over-decolorized, even Gram positive bacteria may appear pink and when under-decolorized gram negative bacteria may appear Gram positive. The Gram reaction also depends on the age of the cell. Old cultures of Gram positive bacteria (where cell walls may be weakened) may readily get decolorized. Gram positive cells affected by cell wall active agents such as lysozyme or antibiotics may become Gram negative. Gram-positive bacteria such Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these cells. In cultures of Bacillus, and Clostridium a decrease in peptidoglycan thickness during cell growth may cause some of them to appear Gram negative. Certain group of bacteria can display variable response to the stain, which can be due to growth stress (e.g., unsuitable nutrients, temperatures, pHs, or electrolytes) that results in a number of nonviable, gram-negative cells in a gram positive culture, but certain bacterial species are known for their gram variability even under optimal growth conditions. Some bacteria tend to appear Gram negative when grown in acidic medium. Loss of cell walls in Gram positive bacteria may render them Gram negative (L-forms). Bacteria totally devoid of cell wall (Mycoplasma) are always Gram negative. Bacteria such as Mycobacterium that have extra waxy content in their cell wall are difficult to stain. Small and slender bacteria such as Treponema, Chlamydia, Rickettsia are often difficult to stain by Gram's method. Gram positive bacteria that have been phagocytosed by polymorphs may also appear Gram negative. 1.6 Structural staining ( not performed)


This type of staining is used to identify particular cellular structures like flagella, spores & capsules ect. 1.7 Isolation of pure cultures of bacteria and fungi from the environment. 1.7.1. Introduction In nature microorganisms exists as mixed populations of many widely different types. If we want to study about a species, we can do it only by separating the species of interest from the mixed population & subsequently growing it in an environment free of contaminations. Pure culture denotes that all cells in the culture has the same origin & are simply descendants of the same cell. This involves the isolation & purification of cultures. Methods of isolation will depend on the source from which the species is isolated. Purification of cultures is commonly done by methods such as Streak plate, Pour plate or spread plate. Once a pure culture has been made it is desirable to maintain c the culture in a viable condition for varying periods of time. For short term preservation &maintenance of cultures we simply transfer the culture to an agar slant stores it in a refrigerator. 1.7.2 Streak Plate Method Most widely used technique for the purification of a bacterial culture is the streak plate method. A small amount of growth is obtained on a sterilized inoculating loop &the loop is then dragged lightly over the surface of the agar in the Petri dish. This method gives isolated bacterial cells at the end of the streak. After a suitable incubation single isolated colonies can be seen along the streak lines.


1.5.3 Spread Plate Method ( Not performed)

Deferent ways of streaking on the agar plate 1.7.3 Materials Sterile Nutrient Agar deeps, sterile Petri dishes, A mixed broth culture of bacteria, Inoculating loop, N/A,PDA slant 1.7.4 Procedure A tube of melted & cooled to 45 – 50 C nutrient agar was poured in to a Petri dish & was allowed to get firmly solidified. Then the inoculating loop was heated to red & cooled before removing the bacterial culture from the tip of the loop. Then the lid of the Petri dish was carefully opened to an extend that only the loop containing culture could put in. the culture was then spread on the agar medium to get isolated colonies. Petri dish was then incubated for 1 to 2 days at 37 C. Observation

Streak plated plates


1.7.5 Discussion This method is used most commonly to isolate pure cultures of bacteria. A small amount of mixed culture is placed on the tip of an inoculation loop/needle and is streaked across the surface of the agar medium. The successive streaks "thin out" the inoculum sufficiently and the microorganisms are separated from each other. It is usually advisable to streak out a second plate by the same loop/needle without reinoculation. In order to identify bacteria, it is necessary to obtain a pure culture. This is done by using the streak-plate method. Bacterial cells are spread over the surface of an agar plate in a continuous dilution, so that the cells will be separated from each other. When the plate is incubated each individual cell will grow in to a colony organized by a single cell. Obtaining well isolated colonies takes practice. There are a variety of techniques used, and each microbiologist obtains his or her own style by experience. Keep the plate covered as much as possible to avoid contamination. While streaking a section of the plate, try to keep the inoculating loop in contact with the surface of the agar at all times. 1.8 Pour plate method This technique gives a second method to obtain a pure culture from a mixed culture. In this method the agar medium is inoculated while it is still in liquid form. Therefore the colonies get developed through out the medium & also on the surface. Better distribution of colonies is obtained in a well made pour plate isolations are more easily made. As there is no accurate way of predicting the number of viable cells in a given sample we need to make several dilutions of the given sample & pour several plates. Sequence of serial dilution Initial volume 10ml 9ml 9ml 9ml 9ml What is added 1g 1ml of 1st 1ml of 2nd 1ml of 3rd 1ml of 4th Dilution factor 10-0 10-1 10-2 10-3 10-4

1 tube 2nd tube 3rd tube 4th tube 5th tube



Thereafter either procedure can be followed 1. 1ml of each dilution is placed in the centre of an empty sterile Petri dish & the molten agar medium which was kept at around 45 C is poured. 2. A loop full of dilutions are transferred in to the tubes of molten agar the inoculums were mixed & the mixture was poured in to separate sterile Petri dishes.

Making a dilution series Colonies grown on agar media cultured by Pour plate technique 1.8.2 Procedure A dilution series was made by transferring 1 ml of the original suspension to the first tube & so on & mixed well by rolling between the palms to ensure the even dispersion of cells through out the suspension.1 ml of each suspension was then ascetically transferred in to the centre of a sterile Petri dish with the help of a sterile pipette. About 15 ml of liquid agar at around 45 C was then transferred to the Petri dish & they were gently mixed by rotating the dish on the table top in clockwise & anticlockwise. It was then allowed to get solidify & incubated at 37 C for 1-2 days inverting them in the incubator. 1.8.3 Results & Observations Number of colonies in 10-2 dilution= 40

CFU/ mL = CFU/plate x dilution factor x 1/aliquot
= 40/10-2 X 1/1 = 40 X102 CFU/ml 8.4 Discussion


This method involves plating of diluted samples mixed with melted agar medium. The main principle is to dilute the inoculum in successive tubes containing liquefied agar medium so as to permit a thorough distribution of bacterial cells within the medium. Here, the mixed culture of bacteria is diluted directly in tubes containing melted agar medium maintained in the liquid state around 45 C. The bacteria and the melted medium are mixed well. The contents of each tube are poured into separate Petri plates, allowed to solidify, and then incubated. When bacterial colonies develop, one finds that isolated colonies develop both within the agar medium (subsurface colonies) and on the medium (surface colonies). These isolated colonies are then picked up by inoculation loop and streaked onto another Petri plate to insure purity. Pour plate method has certain disadvantages as follows: (i) the picking up of subsurface colonies needs digging them out of the agar medium thus interfering with other colonies, and (ii the microbes being isolated must be able to withstand temporary exposure to the 42-45° temperature of the liquid agar medium; therefore this technique proves unsuitable for the isolation of psychrophilic microorganisms. However, the pour plate method, in addition to its use in isolating pure cultures, is also used for determining the number of viable bacterial cells present in a culture.


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