LABORATORY RULES There are some rules which must be observed form the successfulcompletion of the laboratory exercise, personal safety convenienc e of others working in the laboratory. 1) Always wear a laboratory coat or apron before entering a laboratory forpr ote cti ng clo the s for m con tamina tio n or acc ide nta l dis col ora tio n 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 th e even t of pe rs onal in jury such as cut or burn, in fo rm yo ur constructor immediately as bacteria love open wounds. 1
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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.
(microbial polysaccharides), alcohol, sausages, and silage (anima food) are all produced by industrialmicrobiology 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.
Microorganisms are used to produce human or animal biologicals such as insulin, growth hormone, andantibodies. 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 specieswith an annual production of more than two million tons of amino acids, mainly L-glutamate and L-
lysine. 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
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.
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 iscritical 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 Gram-
positive bacteria may lose the stain easily and therefore appear as a mixture of Gram-positive and Gram-
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 onthe 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
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 gram-
negative 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
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 geneticflexibility 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, food-
brewing, meat tenderization, baking, manufacture of Soya products, debittering of protein
hydrolysis’s , 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
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 κ-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 obtainedfrom 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 κ-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 κ-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.
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 10g
2. Peptone 0.5g
3. Agar 1.5g
4. ph 7.2(adjust ph by ph meter)
5. Distilled water 100ml
Petri plate
Inoculating loop
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 30°C 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.