LABORATORY RULES

There are some rules which must be observed form the successful completion of the laboratory exercise, personal safety convenience of others working in the laboratory. 1)Always wear a laboratory coat or apron before entering a laboratory for protecting clothes form contamination or accidental discoloration by staining solution. 2)Before and after each laboratory period clean your workbench with disinfectant like Lysol (1; 500) phenol (1; 100) or 90% ethanol. 3)Keep your laboratory bench clean of everything (e.g. Books, purses, papers, etc.) Except your laboratory equipment and notebook. 4)Never smoke, drink or eat in the laboratory. 5)Never place pencil, labels or any other material in your mouth. 6)If a live cultured spilled, cover the area with a disinfectant such as mercuric chloride for 15 min and then clean it. 7)In the event of personal injury such as cut or burn, inform your constructor immediately as bacteria love open wounds.

8)Long hair should be tied back to minimize contamination of cultures and fire hazards. 9)Be careful of laboratory burners and turn them off when not in use. 10) 11) 12) 13) All microbial cultures should be handled as being potential The waste papers and contaminated glasswares should be kept in Wash your hands with soap and water before and after finishing Broth cultures must never be pipitted with mouth and must be pathogenic. recepecticals provided. work in the laboratory. filled with the use of pipette aids and operated in such a way so as to avoid creating aerosol. 14) 15) 16) 17) Aseptic technique must be rigorously observed at all times. Always culture tubes in an upright position in a rack or basket. You must familiarize yourself in advance with the excise to be Label all the plates tubes, cultures properly before starting an

performed. exercise to be performed.

IMPORTANCE OF MICROBES

Beneficial
Microbes are used to make vaccines (attenuated or weak/dead microbes), which can be injected or inoculated into a host organism to stimulate the production of blood substances (called antibodies) to insure future protection against the possible contact with an unwanted microbe. Vaccines are most often produced by using attenuated viruses or bacteria. Later in the course we will discuss vaccination series such as MMR and DPT. A second important medical use of microbes involves the production of antibiotics like penicillin and bacitracin. Antibiotics are molecules produced by bacteria and molds (or talented chemists) that can be injected, taken orally, or occasionally used topically to inhibit new growth of bacteria. Transferring genes (usually by a loose strand of DNA called a plasmid) cells can be directed to produce needed substances like insulin and erythropoeitin (hormone needed for development of red blood cells). This bio-engineering is an exceptionally important use of microbes in modern science. Students in agronomy (plant technologies) and soil science may know that genes from bacteria like Agrobacterium can be used in bio-engineering to develop disease resistance in plants. Also soil microbes are helpful in breaking down (decomposition) of dead organic material in the soil resulting in addition of nutrients to the soil. Still other soil microbes release minerals that are attached to soil particles and/or fix nitrogen. Since most nitrogen is in the atmosphere and not available to plants, microbes must combine the nitrogen with oxygen to make a soluble molecule called a nitrate which can be taken in through the roots. During the breakdown of carbohydrates by microbes, numerous products like acids and alcohols may be produced. The production of these chemicals is a major industrial use of microbes. In 3

the fermentation process, many foods including cheese and silage (animal food often used in the dairy industry) are the result of microbe activity. Pasteur was an early microbiologist who studied fermentation of grapes resulting in the production of wine. Later he introduced pasteurization (heating) to eliminate additional microbial fermentation so the wine "did not go bad." Saccharomyces (yeast) and Zygomonas (a bacterium) are commonly used to make beer and tequilla respectively. Microbes are also important in environmental studies. The breakdown of sewage and oil slicks (spills) returns the environment to its desired original condition. This return to the original environment is called bio-remediation. In the food chains of nature, microbes are often one of the early sources of energy needed to maintain the community. Microscopie algae convert sunlight into useable food and small protozoans and bacteria serve as a primary (bottom) step in food chains. This microscopic plankton is essential ion water communities.

Industrial microbiology
Industrial microbiology encompasses the use of microorganisms in the manufacture of food or industrial products. The use of microorganisms for the production of food, either human or animal, is often considered a branch of food microbiology. The microorganisms used in industrial processes may be natural isolates, laboratory selected mutants or genetically engineered organisms.

Industrial Application of microbes Food microbiology

Yogurt, cheese, chocolate, butter, pickles, sauerkraut, soya sauce, vitamins, amino acids, food thickeners (microbial polysaccharides), alcohol, sausages, and silage (anima food) are all produced by industrial microbiology processes. "Good" bacteria such as probiotics are becoming increasingly important in the food industry.

Biopolymers
A huge variety of biopolymers, such as polysaccharides, polyesters, and polyamides, are produced by microorganisms. These products range from viscous solutions to plastics. The genetic manipulation of microorganisms has permitted the biotechnological production of biopolymers with tailored material properties suitable for high-value medical application such as tissue engineering and drug delivery. Industrial microbiology can be used for the biosynthesis of xanthan, alginate, cellulose, cyanophycin, poly(gamma-glutamic acid), levan, hyaluronic acid, organic acids, oligosaccharides and polysaccharides, and polyhydroxyalkanoates.

Bioremediation
Microbial biodegradation of pollutants can be used to cleanup contaminated environments. These bioremediation and biotransformation methods harness naturally occurring microbes to degrade, transform or accumulate a huge range of compounds including hydrocarbons (e.g. oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), pharmaceutical substances, radionuclides and metals.

Waste biotreatment
Microorganisms are used to treat the vast quantities of wastes generated by modern societies. Biotreatment, the processing of wastes using living organisms, is an environmentally friendly, relatively simple and cost-effective alternative to physico-chemical clean-up options. Confined environments, such as bioreactors] can be employed in biotreatment processes. 5

Health-care and medicine

Microorganisms are used to produce human or animal biologicals such as insulin, growth hormone, and antibodies. Diagnostic assays that use monoclonal antibody, DNA probe technology or real-time PCR are used as rapid tests for pathogenic organisms in the clinical laborarory.

Archaea
Examination of microbes living in unusual environments (e.g. high temperatures, salt, low pH or temperature, high radiation) an lead to discovery of microbes with new abilities that can be harnessed for industrial purposes.

Corynebacteria
Corynebacteria are a diverse group Gram-positive bacteria found in a range of different ecological niches such as soil, vegetables, sewage, skin, and cheese smear. Corynebacterium glutamicum is of immense industrial importance and is one of the biotechnologically most important bacterial species with an annual production of more than two million tons of amino acids, mainly L-glutamate and Llysine. The genome sequence of C. glutamicum has been published

Harmful
Earlier we stated that most microbes are beneficial; however, some microbes do damage and are potentially harmful and costly. Some microbes cause disease and are called pathogens. Early microbiologists like Louis Pasteur and Robert Koch completed experiments showing that diseases could arise when microbes entered and grew in tissues. Between 1875 and 1900 Robert Koch found the causative microbes of at least 20 diseases. This germ theory of disease, which identifies the cause of disease- one microbe, one disease, is still used and important today. Large populations of microbes may also cause infestations thus reducing the potential productivity of host organisms. Examples of microbes that infest other living organisms include roundworms or nematodes, flukes, tapeworms, and spider mites. These ectoparasites (on surface) and endoparasites (inside host) may infest as many as 50 million people annually in our country. If bacteria increase in high numbers in water, oxygen is depleted and many living organisms like fish may die form lack of oxygen. 6

The presence of microbes also may indicate spoilage. It is estimated that 30% or more of all food produced in our country may be lost to spoilage. The change in color, texture, or smell of the food makes the food less palatable and therefore is often discarded. Spoilage of food is defined as food discarded because of the presence of microbes and/or toxins (poisons) released in to the food.

Isolation and enumerationof Bacteria form Soil Sample

PURPOSE

Isolation and enumeration of microorganisms from soil sample .

MATERIALS
Cotton swabs, nutrient agar media, 3 petri plates, 6.test tubes, magnetic shaker, pipette, distelled water, colony counter, 1.conical flask.

COMPOSITION OF NUTRIENT AGAR. (100 ML)

Agar 2g Distelled water 100ml NaCl 5g Beef extract 3g Peptone ------ .5g

METHOD USED-- SERIAL DILUTION AGAR-PLATING METHOD

FORMULA USED No of cells per g of soil = (mean plate count *dilution factor)/dry wt of soil.

PRINCIPLE NO of colonies developed = no of microorganism in the media

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EXPERIMENTAL PROCEDURE
10ml sterile distilled water was taken in a test tube. Take 1gm of soil sample and dissolve in the first test tube.. The tube was vigorously vortexed for 3 minutes to obtain uniform suspension of microorganisms. A series of tubes, labeled as 10-1, 10-2 to 10-7, were filled with 9ml sterile distilled water. 1ml of diluted sample was transferred to the tube marked 10-1 and vigorously vortexed. 1ml from this tube was transferred to the tube marked as 10-2 and vigorously vortexed. The process was repeated up to 10-7. Pour plate method was used to culture Bacteria using Nutrient Agar media. Three sterile Petri plates were taken and labeled as 10-2, 10-3, and 10-4. To this 1 ml aliquots each from 10-2, 10-3, and 10-4 serial dilution were transferred to respective pertiplates. Then approximately 15ml of cooled (450 C) starch agar medium was added to each Petri plate and the inoculums was mixed by gentle rotation of Petri plates. After solidification of medium the plates were incubated for 24 hours at 370C.

OBSERVATION
After incubation bacterial colonies were developed in all the Petri plates. First two Petri plates were highly covered with bacterial colonies so unable to count. Third Petri plate contains countable number of colonies.

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 Number of colonies counted were = 71

VIABLE COUNT
10-1 dilution:

10-2 dilution:

10-3 dilution:

10-4 dilution:

10-5 dilution:

10-6 dilution:

ENUMERATION OF MICROORGANISMS BY VIABLE COUNT

11

CALCULATION No of cells per g of soil = (mean plate count *dilution factor)/dry wt of soil = ( 71 * 10^6)/0.8 = RESULT The number of cells per gram of soil=

12

TECHNIQUE FOR ISOLATION OF PURE CULTURES:

Mature, microbial populations do not segregate themselves by species but exist with a mixture of any other cell types. In the laboratory, these populations can be separated into pure cultures. These cultures contain only one type of organism which is suitable for the study of their cultural, morphological, and biochemical properties. In this experiment, technique is designed to produce discrete colonies. Colonies are

individual, macroscopically visible masses of microbial growth on a solid medium surface, each representing the multiplication of a single organism. Once stained these discrete colonies, we will make aseptic transfer onto nutrient agar slants for isolation of pure cultures.

AIM
Perform the spread-plate and/or the streak-plate inoculation procedure for the separation of the cells of a mixed culture so that discrete colonies can be isolated.

PRINCIPLE
The techniques commonly used for isolation of discrete colonies initially require that the number organisms in the inoculum be reduced. The diminution of the population size ensures that, following inoculation, individual cell will be sufficiently far apart on the surface of the agar medium to effect a separation of the different species present. The following are techniques that can be used to accomplish this necessary dilution. The streak-plate method is a rapid qualitative isolation method. It is essentially a dilution technique that involves spreading a loopful of culture over the surface of an agar plate. Although many types of procedures are performed, the four-way, or quadrant, streak is described.

PROCEDURE:
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Place a loopful of culture on the agar surface in Area 1. Flame and cool the loop and drag it rapidly several times across the surface of Area 1. Reflame and cool the loop and turn the Petri dish 90. Then touch the loop to a corner of the culture in Area 1 and drag it several times across the agar in Area 2. The loop should never enter Area 1 again.

Reflame and cool the loop and again turn the dish 90. Streak Area 3 in the same manner as Area 2. Without reflaming the loop, again turn the dish 90 and then drag the culture from a corner of Area 3 across Area 4, using a wider streak. Dont let the loop touch any of the previously streaked areas. The flaming of the loop at the points indicated is to effect the dilution of the culture so that fewer organisms are streaked in each area, resulting In the final desired separation. The spread-plate technique requires that a previously diluted mixture of microorganisms be used.

During inoculation, the cells are spread over the surface of a solid agar medium with a sterile, L-shaped bent rod while the Petri dish is spun on a lazy-Susan turntable. The step-by-step procedure for this technique is as follows: Place the bent glass rod into the beaker and add a sufficient amount of 95% ethyl alcohol to cover the lower, bent portion. With a sterile loop, place a loopful of M. luteus culture in the center of the appropriately labeled nutrient agar plate that has been placed on the turntable. Replace the cover. Remove the glass rod from the beaker and pass it through the Bunsen burner flame, with the bent portion of the rod pointing downward to prevent the burning alcohol from running down your arm. Allow the alcohol to burn off the rod completely. Cool the rod for 10 to 15 seconds. While the Petri dish cover and spin the turntable. While the sterile bent rod to the surface of the agar and move it back and forth. This will spread the culture over the agar surface. When the turntable comes to a stop, replace the cover. Immerse the rod in alcohol and reflame. The pour-plate technique requires a serial dilution of the mixed culture by means of a loop or pipette. The diluted inoculum is then added to a molten agar medium in a Petri dish, mixed, and allowed to solidify.

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BACTERIAL STAINING
WHAT ARE BACTERIA?
Until recently, the term bacteria were used for all microscopic prokaryotes. But, it turns out that there are two groups of prokaryotes that differ from each other in just about every way except size and lack of a nucleus. These are now distinguished as the:

Bacteria; the "true" bacteria (also known as Eubacteria) Archaea; (also known as Archaebacteria)

The archaea are so different from the bacteria that they must have had a long, independent evolutionary history since close to the dawn of life. In fact, there is considerable evidence that you are more closely related to the archaea than they are to the bacteria!

PROPERTIES

Prokaryotic (no membrane-enclosed nucleus) No mitochondria or chloroplasts A single chromosome

o o

A closed circle of double-stranded DNA With no associated histones

If flagella are present, they are made of a single filament of the protein flagellin; there are none of the "9+2" tubulin-containing microtubules of the eukaryotes. Ribosomes differ in their structure from those of eukaryotes Have a rigid cell wall made of peptidoglycan. The plasma membrane is a phospholipid bilayer but contains no cholesterol or other steroids. no mitosis Mostly asexual reproduction Any sexual reproduction very different from that of eukaryotes; no meiosis

15

Many bacteria form a single spore when their food supply runs low. Most of the water is removed from the spore and metabolism ceases. Spores are so resistant to adverse conditions of dryness and temperature that they may remain viable even after 50 years of dormancy.

CLASSIFICATION
Until recently classification has done on the basis of such traits as:

Shape
o o o

Bacilli: rod-shaped Cocci: spherical Spirulla: curved walls

Ability to form spores Method of energy production (glycolysis for anaerobes, cellular respiration for aerobes) Nutritional requirements Reaction to the Gram stain.

Although the Gram stain might seem an arbitrary criterion to use in bacterial taxonomy, it does, in fact, distinguish between two fundamentally different kinds of bacterial cell walls and reflects a natural division among the bacteria. More recently, genome sequencing, especially of their 16S ribosomal RNA (rRNA), has provided new insights into the evolutionary relationships among the bacteria.

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GRAM STAIN
HISTORY AND HOW IT WORKS
The Gram staining method, named after the Danish bacteriologist who originally devised it in 1844, 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 violet-iodine 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 non-negative. Others that are not stained by crystal violet are referred to as Gram negative, and appear red. 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 violet-iodine 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 Gramnegative bacteria (Gram-variable).

17

Finally, a counterstain of basic fuchsin is applied to the smear to give decolorized gram-negative bacteria a pink color. Some laboratories use safranin as a counterstain 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. Besides Gram's stain, there are a wide range of other staining methods available. By using appropriate dyes, different parts of the bacteria structures such as capsules, flagella, granules, and spores can be stained. Staining techniques are widely used to visualize those components that are otherwise too difficult to see under a light microscope. In addition, special stains can be used to visualize other microorganisms not readily visualized by the Gram stain, such as mycobacteria, rickettsia, spirochetes, and others. In addition, there are modifications of the Gram stain that allow morphologic analysis of eukaryotic cells in clinical specimens.

AIM
To become familiar with The chemical and theoretical bases for differential staining procedures. The chemical basis of the gram stain. Performance of the procedure for differentiating between the two principal groups of bacteria: gram-positive and gram-negative.

PRINCIPLE
Differential staining requires the use of at least three chemical reagents that are applied sequentially to a heat-fixed smear. The first reagent is called the primary stain. Its function is to impart its color to all cells. In order to establish a color contrast, the second reagent used is the decolorizing agent. Based on the chemical composition of cellular components, the decolorizing agent may or may not remove the primary stain from the entire cell or only from certain cell structures. The final reagent, the counterstain, has a contrasting color to that of the primary stain. Following decolorization, if the primary stain is not washed out, the counterstain cannot be absorbed and the cell or its components will retain the color of the primary stain. If the primary stain is removed, the decolorized cellular 18

components will accept and assume the contrasting color of the counterstain. In this way, cell types or their structures can be distinguished from each other on the basis of the stain that is retained. The most important differential stain used in bacteriology is the Gram stain, named after Dr. Christian Gram. It divides bacterial cells into two major groups, gram-positive and gram-negative, which makes it an essential tool for classification and differentiation of microorganisms. The Gram stain uses four different reagents. Descriptions of these reagents and their mechanisms of action follow.

PRIMARY STAIN
Crystal Violet, this violet stain is used first and stains all cells purple.

MORDANT
Grams Iodine serves as a substance that forms an insoluble complex by binding to the primary stain. The resultant crystal violet-iodine (CV-I) complex serves to intensify the color of the stain, and all the cells will appear purple-black at this point. In gram-positive cells only, this CV-I complex binds to the magnesium-ribonucleic acid component of the cell wall. The resultant magnesium-ribonucleic acidcrystal violet-iodine (Mg-RNA-CV-I) complex is more difficult to remove than the smaller CV-I complex.

DECOLORIZING AGENT
Ethyl Alcohol, 95% serves a dual function as a lipid solvent and as a protein-dehydrating agent. Its action is determined by the lipid concentration of the microbial cell walls. In gram-positive cells, the low lipid concentration is important to retention of the Mg-RNA-CV-I complex. Therefore, the small amount of lipid content is readily dissolved by the action of the alcohol, causing formation of minute cell wall pores. These are then closed by alcohols dehydrating effect. As a consequence, the tightly bound primary stain is difficult to remove, and the cells remain purple. In gram-negative cells, the high lipid concentration found in outer layers of the cell wall is dissolved by the alcohol, creating large pores in the cell wall that do not close appreciably on dehydration of cell wall proteins. This facilitates release of the unbound CV-I complex, leaving these cells colorless or unstained.

19

COUNTER STAIN
Safranin is the final reagent, used to stain red those cells that have been previously decolorized. Since only gram-negative cells undergo decolorization, they may now absorb the counterstain. Gram-positive cells retain the purple color of the primary stain.

The preparation of adequately stained smears requires that you bear in mind the following precautions:
1. The most critical phase of the procedure is the decolorization step, which is based on the ease with which the CV-I complex is released from the cell. Remember that over-decolorization will result in loss of the primary stain, causing gram-positive organisms to appear gram-negative. Under-decolorization, however, will not completely remove the CV-I complex, causing gramnegative organisms to appear gram-positive. Strict adherence to all instructions will help remedy part of the difficulty, but individual experience and practice are the keys to correct decolorization.

2. It is imperative that slides be thoroughly washed under running tap water between applications of the reagents. This removes excess reagent and prepares the slide for application of the subsequent reagent.

3. The best gram stained preparations are made with fresh cultures, that is, not older than 24 hours. As cultures age, especially in the case of gram-positive cells, the organisms tend to lose their ability to retain the primary stain and may appear to be gram-variable; that is, some cells will appear purple, while others will appear red.

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DIAGRAM

GRAM STAINING

MATERIALS CULTURES
24-hour nutrient agar slant cultures of microorganisms obtained from the soil sample(E.coli,Bacillus, Proteus vulgaris,Pseudonomas).

21

REAGENTS
Crystal violet, Grams iodine, 95%ethyl alcohol, and Safranin.

EQUIPMENTS
Bunsen burner, inoculating loop or needle, staining tray, glass slides, bibulous paper, lens paper and microscope.

PROCEDURE
The steps are as follows; 1. Obtain four clean glass slides. 2. Using sterile technique, prepare a smear of each of the three organisms and on the remaining slide prepare a smear consisting of a mixture of S.aureus and E.coli. Do this by placing a drop of water on the slide and then transferring each organism separately to the drop of water on the slide with a sterile, cooled loop. Mix and spread both organisms by means of a circular motion of the inoculating loop. 3. Allow smears to air dry and then heat fix in the usual manner. 4. Flood smears with crystal violet and let stand for 1 minute. 5. Wash with tap water. 6. Flood smears with the grams iodine mordant and let stand for 1 minute. 7. Wash with tap water. 8. Decolorize with 95% ethyl alcohol. Caution: do not over decolorize. Add reagent drop by drop until crystal violet fails to wash from smear. 9. Wash with tap water. 10. Counterstain with Safranin for 45 seconds. 11. Wash with tap water. 12. Blot dry with bibulous paper and examine under oil immersion.

22

OBSERVATION
1st slide appears red so gram negative bacteria.(E.coli). 2nd slide appears blue so gram positive bacteria(Bacillus). 3rd slide appears red so gram negative bacteria.(Proteus vulgaris) 4th slide appears red so gram negative bacteria.(Pseudomonas)

Typical Gram stain

GRAM POSITIVE

GRAM NEGATIVE

RESULT
Both gram(+&-) bacteria are obtained in the soil sample.

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ENDOSPORE STAINING
THEORY
The endospore stain is a differential stain used to visualize bacterial endospores. Endospores are formed by a few genera of bacteria, such as Bacillus. By forming spores, bacteria can survive in hostile conditions. Spores are resistant to heat, dessication, chemicals, and radiation. Bacteria can form endospores in approximately 6 to 8 hours after being exposed to adverse conditions. The normallygrowing cell that forms the endospore is called a vegetative cell. Spores are metabolically inactive and dehydrated. They can remain viable for thousands of years. When spores are exposed to favorable conditions, they can germinate into a vegetative cell within 90 minutes. Endospores can form within different areas of the vegetative cell. They can be central, subterminal, or terminal. Central endospores are located within the middle of the vegetative cell. Terminal endospores are located at the end of the vegetative cell. Subterminal endospores are located between the middle and the end of the cell. Endospores can also be larger or smaller in diameter than the vegetative cell. Those that are larger in diameter will produce an area of swelling in the vegetative cell. These endospore characteristics are consistent within the spore-forming species and can be used to identify the organism. Because of their tough protein coats made of keratin, spores are highly resistant to normal staining procedures. The primary stain in the endospore stain procedure, malachite green, is driven into the cells with heat. Since malachite green is watersoluble and does not adhere well to the cell, and since the vegetative cells have been disrupted by heat, the malachite green rinses easily from the vegetative cells, allowing them to readily take up the counterstain.

AIM- To identify the endospore forming bacteria in the soil sample.

24

REQUIRMENTSMalachite green, safarine, staining tray, glass slide,pure cultures. (E.coli,Bacillus, Proteus vulgaris,Pseudonomas).

PROCEDURE
1. Make smears of the 5 pure samples on the slide. 2. Steam for 5 min and add malachite green time to time. 3. Wash under running water. 4. Blot dry the slides 5. Add safrenin to it keep for 10 min 6. Wash it.

OBSERVATION
If colour comes to green then it is endospore former otherwise if the colour comes non green then it is non endospore former. 1ST slide is not green therefore non endospore former bacteria.(E.coli) 2nd slide is green therefore endospore former bacteria.(Bacillus) 3rd slide is not green therefore non endospore former bacteria.(Proteus vulgaris) 4th slide is not green therefore non endospore former bacteria.(Pseudomonas)

DIAGRAM
25

ENDOSPORE FORMING BACTERIA

RESULT
Both endospore former and non former bacteria obtained fro the soil sample

26

SCREENING OF MICROORGANISMS FOR INDUSTERIAL PURPOSE

AIM: Screening of proteases (caseinase) producing bacteria. THEORY

Proteases are the single class of enzymes, which occupy a pivotal position with respect to their applications in both physiological and commercial fields. They perform both degradative and synthetic functions. Proteolytic enzymes catalyze the cleavage of peptide bonds in other proteins. Advances in analytical technique have demonstrated that proteases conduct highly specific and selective modifications of proteins. Proteases are classified on the basis if three major criteria: (1) Type of reaction catalyzed (2) Chemical nature of the catalytic site and (3) Evolutionary relationship with reference to structure. Proteases are grossly subdivided into two major groups i.e., exopeptides and endopeptides, depending on their site of action. Based on the functional group present at the site active site, proteases are further classified into 4 prominent groups i.e. serine proteases, cysteine proteases, aspartic proteases and metallo proteases. Proteases are also divided into acid, neutral and alkaline proteases on the basis of pH range in which their activity is optimum.

27

Proteases occur ubiquitously in a wide diversity of sources such as plants, animals and microorganisms. Microbes are the attractive sources of proteases and have gained much popularity than any other sources because of their broad biochemical diversity. The inability of the plant and animal proteases to meet current world demands has lead to an increased interest in microbial proteases. Microbial enzymes have two advantages over the animal and plant enzyme. Firstly, they are economical and can be produced on a large scale within the limited space and time. It can be easily extracted and purified. Secondly, there is a technical advantage in producing enzymes via using micro organisms as (1) they are capable of producing wide variety of enzymes (2) they show genetic flexibility that is why they can be genetically manipulated to increase the yield of enzymes and they have a short generation time. Proteases execute a large variety of functions, extending from the cellular level to the organ and organism level. Their involvement in the life cycle of disease organisms has lead them to become a potential target for developing therapeutic agents against fatal disease such as cancer and AIDS. Proteases have a long history of applications in different industries viz, detergents, foodbrewing, meat tenderization, baking, manufacture of Soya products, debittering of protein hydrolysiss , synthesis of aspartame, dairy, leather, silk and for recovery of silver from used x-ray films. Besides their industrial and medicinal applications, proteases play an important role in basic research. Their selective peptide bond cleavage is used in the study of sequencing of proteins. A wide range of microorganisms including bacteria, yeast and also mammalian tissues produces alkaline proteases.

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CASEIN
Casein (from Latin caseus "cheese") is the predominant phosphoprotein.that account for nearly 80% of proteins in milk and cheese. Milk-clotting proteases act on the soluble portion of the caseins, K-Casein, thus originating an unstable micellar state that results in clot formation. When coagulated with rennet, casein is sometimes called paracasein. Chymosin (EC 3.4.23.4) is an aspartic protease that specifically hydrolyzes the peptide bond in Phe105-Met106 of -casein and is considered to be the most efficient protease for the cheese-making industry.British terminology, on the other hand, uses the term caseinogen for the uncoagulated protein and casein for the coagulated protein. As it exists in milk, it is a salt of calcium. Casein is not coagulated by heat. It is precipitated by acids and by rennet enzymes, a proteolytic enzyme typically obtained from the stomachs of calves. The enzyme trypsin can hydrolyze off a phosphate-containing peptone. Casein consists of a fairly high number of proline peptides, which do not interact. There are also no disulfide bridges. As a result, it has relatively little secondary structure or tertiary structure. Because of this, it cannot denature. It is relatively hydrophobic, making it poorly soluble in water. It is found in milk as a suspension of particles called casein micelles which show some resemblance with surfactant-type micellae in a sense that the hydrophilic parts reside at the surface. The caseins in the micelles are held together by calcium ions and hydrophobic interactions. There are several models that account for the special conformation of casein in the micelles. One of them proposes that the micellar nucleus is formed by several submicelles, the periphery consisting of microvellosities of -casein. Another model suggests that the nucleus is formed by casein-interlinked fibrils Finally, the most recent model proposes a double link among the caseins for gelling to take place. All 3 models consider micelles as colloidal particles formed by casein aggregates wrapped up in soluble -casein molecules. The isoelectric point of casein is 4.6. The purified protein is water insoluble. While it is also insoluble in neutral salt solutions, it is readily dispersible in dilute alkalis and in salt solutions such as sodium oxalate and sodium acetate.

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PRINCIPLE:
It is based on the principle that some microorganisms have the ability to degrade the protein, casein by producing proteolytic exoenzymes, proteinase or caseinase, which breaks the peptide bond CO-NH into free amino acids.

REQUIRMENTS:
Slant culture of test organism(E.COLI) Skimmed milk agar. or casein media 1. Skim milk powder 2. Peptone 3. Agar 4. ph 5. Distilled water Petri plate Inoculating loop 10g 0.5g 1.5g 7.2(adjust ph by ph meter) 100ml

PROCEDURE:
The autoclaved skimmed milk medium was poured into sterile plate and allowed to solidify .Upon solidification of the medium single round streak inoculation was done from each isolate at the two sides of the plate. The plate was incubated at 30C for 24-48 hours in an inverted position. After incubation the plate was observed for any clear zones around the growth of the organisms. Mass production of the enzyme can be done either by submerged fermentation or solid substrate fermentation. In submerged fermentation, the organism was cultivated in liquid media in the flasks for the enzyme production whereas in the solid substrate fermentation, the culture was inoculated across the surface of production medium and the culture remains on the surface through out the fermentation.

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OBSERVATION:
A clear zones around the growth of the organisms is observed.

DIAGRAM:

CASEIN DEGRADATION
RESULT:
Screening of proteases producing microorganism done.

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AIM: Screening of Amylase producing bacteria by starch hydrolysis. THEORY

The ability to degrade starch is used as a criterion for the determination of amylase production by a microbe. In the laboratory it is tested by performing the starch test to determine the absence or presence of starch in the medium by using iodine vapor as an indicator. Starch in the presence of iodine produces a dark blue coloration of the medium around the colony.

PRINCIPLE:
The extracellular enzyme alpha amylase catalyzes the breakdown of starch to maltose. The smaller maltose molecules can enter the cell to be used for energy. Iodine interacts with starch and makes it dark blue. If starch has been removed from a culture medium by the action of the bacterial enzyme no blue color appears in the presence of iodine. If iodine stains the starch medium blue in the presence of growing bacterial colonies then no amylase is produced by that species

REQUIRMENTS
Starch agar media 1. Starch 2. Peptone 3. Beef extract 4. Agar 5. dDstilled water Petriplates Previously cultured media(bacillus culture) Inoculating loop 2g 0.5g 0.3g 1.5g 100 ml

PROCEDURE
The starch agar medium was melted, cooled to 450C and poured into the sterile Petri dish. Allowed it to solidify, 32

Using sterile techniques a single streak inoculation of Bacillus subtilis was made at the center of the Petri plate. The Petri plates were incubated for 72 hours at room temperature. A Luxurious growth of Bacillus subtilis was seen along the streak. The plates were exposed to Iodine vapors.or iodinr added to them.

OBSERVATION
The zone of clearing surrounding Bacillus indicate that it was able to hydrolyze starch. Due to which a clear sharp boundry of transparent type is developed.

DIAGRAM

STARCH DEGRADATION SHOWING AMYLASE PRODUCTION

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CLOSE UP
RESULT:
Screening of Amylase producing bacteria by starch hydrolysis done.

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REFERENCES

1. Madigan M, Martinko J (editors) (2006). Brock Biology of Microorganisms, 11th ed., Prentice Hall. ISBN 0-13-144329-1. 2. Rice G (2007-03-27). Are Viruses Alive?. Retrieved on 2007-07-23. 3. Amann RI, Ludwig W, Schleifer KH (1995). "Phylogenetic identification and in situ detection of individual microbial cells without cultivation". Microbiol. Rev. 59: 143-169. 4. Varro On Agriculture 1,xii Loeb 5. Ibrahim B. Syed, Ph.D. (2002). "Islamic Medicine: 1000 years ahead of its times", Journal of the Islamic Medical Association 2, p. 2-9. 6. David W. Tschanz, MSPH, PhD (August 2003). "Arab Roots of European Medicine", Heart Views 4 (2). 7. Gest H (2005). "The remarkable vision of Robert Hooke (1635-1703): first observer of the microbial world". Perspect. Biol. Med. 48 (2): 266-72. 8. doi:10.1353/pbm.2005.0053. PMID 15834198. 9. Drews G (1999). "Ferdinand Cohn, a Founder of Modern Microbiology". ASM News 65 (8). 10.Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology, 4th ed., McGraw Hill. ISBN 0-8385-8529-9.

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