EXPERIMENT1-A.docx

DATE : 17TH OCTOBER 2009

TIME : 8.00 A.M

TITTLE : BACTERIA AND FUNGI

A.STAINING BACTERIA

Simple Staining

OBJECTIVE ;-

1) Differentiate stain uses contrasting stains to colour different structures.

2) Separate the bacteria into different groups.

3) To study the general simple method of staining bacteria cells using crystal violet dye.

MATERIALS ;-

1) Bacteria cultures Escherichia coli (-ve) and Straphylococus eperdermidos (+ve).

2) Glass slides (at least 2).

3) Bunsen burner

4) Crystal violet crystals (0.5% w/v)

5) Staining rack

6) Microscope (with 10x, 40x and 100x objectives)

7) Filter paper

8) Wire loop

9) Sterile water (squirt bottle)

10) Detergent

UNIVERSITI SAINS MALAYSIA _____________________________________________________________________

METHOD ;-

1) Preparing a bacterial smear ;

- Use detergent to clean a glass slide, after rinse with water dry it.

- To sterile it, heat a wire loop.

- Transfer one small drop of sterile water to center of a a glass slide by using loop.

- Sterile the loop and let it cool for a while.

- From a Culture Escherichia Coli, touch a single colony of bacteria.

- Mix well after transferred the bacteria to the water droplet on the slide.

- Allow the bacterial slurry to air dry.

- Hold the dry slide by one edge and pass it slowly through a Bunsen Burner flame three times,

which is sufficient to kill the bacteria and cause them to adhere.

2) Staining process ;

- Using the crystal violet stain, cover the smear after the bacterial smear slide have been placed

in a staining rack.

- In 30 seconds to 1 minute allow the stain to work.

- By rinse with water, remove the stain.

- Gently blot the stain dry using filter paper.

- By using microscope, it is ready to take a look to the slide.

3) Microscope examination ;

- Before putting the slide on the microscope, it is must be completely dry.

- To locate a good view field, firstly need to observe it with low power(10x objective).

- Examine with 40x objective after successfully located the bacterial cells.

- Focus onto cells which are clearly stained.

- Add a drrop of immersion oil and swing the 100x oil immersion objective lens into the oil. Use

only fine focus to bring image into focus.


RESULT;-

1) Diagram of Bacteria

QUESTION ;-

1) Describe the bacteria that you see in your preparation (form, size, single vs.aggregates).UNIVERSITI SAINS MALAYSIA _____________________________________________________________________

ANSWER ;-

1) Although all bacteria share certain structural, genetic, and metabolic characteristics, important biochemical differences exist among the many species of bacteria. These differences permit bacteria to live in many different, and sometimes extreme, environments. Bacteria are mostly unicellular organisms that lack chlorophyll and are among the smallest living things on earth—only viruses are smaller. Multiplying rapidly under favorable conditions, bacteria can aggregate into colonies of millions or even billions of organisms within a space as small as a drop of water.

The lowest temperature at which a particular species will grow is the minimum growth temperature, while the maximum growth temperature is the highest temperature at which they will grow. The temperature at which their growth is optimal is called the optimum growth temperature. In general, the maximum and minimum growth temperatures of any particular type of bacteria are about 30°F (-1°C) apart. Like temperature, pH also plays a role in determining the ability of bacteria to grow or thrive in particular environments. Most commonly, bacteria grow optimally within a narrow range of pH between 6.7 and 7.5.

Like temperature, pH also plays a role in determining the ability of bacteria to grow or thrive in particular environments. Most commonly, bacteria grow optimally within a narrow range of pH between 6.7 and 7.5.

CONCLUSION ;-


Staining provides a reliable means for observing bacteria in terms of their relative size, morphology and cellular arrangement. Stains are solutions of a dye that has been dissolved in water or alcohol. The dye is a salt (like Na+Cl-). One component of the dye consists of a negatively charged ion, while the other component of the cell consists of a positively charged ion. The ionic component of the dye that imparts color to the cell is called a chromophore. In the case of basic dyes the chromophore is positively charged. The positively charged component of a basic dye is able to interact strongly with a bacterial cell, because most cells under normal conditions have an overall negative charge. The stains that we will be using in the following exercises, methylene blue, crystal violet, malachite green and safranin (red) are all examples of basic dyes.

Differential Staining : Gram Staining


OBJECTIVE ;-

1) To carry out the Gram Stain on Bacteria cultures Escherichia coli (-ve) and Straphylococus

eperdermidos (+ve).

2) Determine their reactions towards the stain.

3) To study the four basic steps of the Gram Stain, which include applying a primary stain

(crystal violet) to a heat-fixed smear of a bacterial culture.

MATERIALS ;-

1) Bacteria cultures A and B

2) Glass slide

3) Bunsen burner

4) Crystal violet crystals (0.5% w/v) – the primary stain

5) Iodine solution (IKI) - the mordant

6) Ethyl alcohol (95%) – the decoloriser

7) Safranin – the counterstain

8) Staining rack

9) Microscope (with 10x, 40x, and 100x)

10) Filter paper

11) Wire loop

12) Sterile water – preferably in a squirt bottle

13) Detergent

METHOD ;-


1) Preparing a bacterial smear ;

- Use detergent to clean a glass slide, after rinse with water dry it.

- To sterile it, heat a wire loop.

- Transfer one small drop of sterile water to center of a a glass slide by using loop.

- Sterile the loop and let it cool for a while.

- From a Culture A, touch a single colony of bacteria.

RESULT;-


1) Labelled of microscope used in this experiment

QUESTION ;-

1) Can you observe the bacteria cell structures just by using light microscope? Why?


2) These questions are related to Gram Staining ;-

a) Why are cells stained with iodine solution?

b) Why do you need to rinse with ethyl alcohol?

c) Why are cells counter cells stained with safranin? Name other counter stains.

d) Why do you need to use young bacteria cultures (24-48h) for this Gram test?

e) Discuss briefly the mechanism of Gram staining and the result expected.

ANSWER ;-


1)Bacteria are microscopic organisms whose single cells have neither a membrane-

bounded nucleus nor other membrane-bounded organelles like mitochondria and chloroplasts. Another group of microbes, the archaea, meet these criteria but are so different from the bacteria in other ways that they must have had a long, independent evolutionary history since close to the dawn of life. Bacteria are microscopic organisms whose single cells have neither a membrane-bounded nucleus nor other membrane-bounded organelles like mitochondria and chloroplasts. Another group of microbes, the archaea, meet these criteria but are so different from the bacteria in other ways 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.

2) (a) Cell staining is a technique that can be used to better visualize cells and cell components under a microscope. By using different stains, one can preferentially stain certain cell components, such as a nucleus or a cell wall, or the entire cell. Most stains


can be used on fixed, or non-living cells, while only some can be used on living cells; some stains can be used on either living or non-living cells. The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample. Cells may also be enumerated by staining cells to determine biomass in an environment of interest.

(b) Ethyl alcohol is an important industrial chemical; it is used as a solvent, in the synthesis of other organic chemicals, and as an additive to automotive gasoline (forming a mixture known as a gasohol). Ethyl alcohol is also the intoxicating ingredient of many alcoholic beverages such as beer, wine, and distilled spirits. There are two main processes for the manufacture of ethyl alcohol: the fermentation of carbohydrates (the method used for alcoholic beverages) and the hydration of ethylene. Fermentation involves the transformation of carbohydrates to ethyl alcohol by growing yeast cells. The chief raw materials fermented for the production of industrial alcohol are sugar crops such as beets and sugarcane and grain crops such as corn (maize). Hydration of ethylene is achieved by passing a mixture of ethylene and a large excess of steam at high temperature and pressure over an acidic catalyst.

Ethyl alcohol produced either by fermentation or by synthesis is obtained as a dilute aqueous solution and must be concentrated by fractional distillation. Direct distillation can yield at best the constant-boiling-point mixture containing 95.6 percent by weight of ethyl alcohol. Dehydration of the constant-boiling-point mixture yields anhydrous, or absolute, alcohol. Ethyl alcohol intended for industrial use is usually denatured (rendered unfit to drink), typically with methanol, benzene, or kerosene.

Pure ethyl alcohol is a colourless, flammable liquid (boiling point 78.5 °C [173.3 °F]) with an agreeable ethereal odour and a burning taste. Ethyl alcohol is toxic, affecting the central nervous system. Moderate amounts relax the muscles and produce an apparent stimulating effect by depressing the inhibitory activities of the brain, but larger amounts impair coordination and judgment, finally producing coma and death. It is an addictive drug for some persons, leading to the disease alcoholism.

Ethyl alcohol is converted in the body first to acetaldehyde and then to carbon dioxide and water, at the rate of about half a fluid ounce, or 15 ml, per hour; this quantity corresponds to a dietary intake of about 100 calories.

c) Safranin is a biological stain used in histology and cytology. Safranin is used as a counterstain in some staining protocols, colouring all cell nuclei red. This is the classic


counterstain in a Gram stain. It can also be used for the detection of cartilage, mucin and mast cell granules.

Safranin typically has the chemical structure shown at right. There is also trimethyl safranin, which has an added methyl group in the ortho- position of the lower ring. Both compounds behave essentially identically in biological staining applications, and most manufacturers of safranin don't distinguish between the two. Commercial safranin preparations often contain a blend of both types. Safranin is also used as redox indicator in analytical chemistry.

Safranines are the azonium compounds of symmetrical 2,8-dimethyl-3,7-diamino-phenazine. They are obtained by the joint oxidation of one molecule of a para-diamine with two molecules of a primary amine; by the condensation of para-aminoazo compounds with primary amines, and by the action of para-nitrosodialkylanilines with secondary bases such as diphenylmetaphenylenediamine. They are crystalline solids showing a characteristic green metallic luster; they are readily soluble in water and dye blue or violet. They are strong bases and form stable monacid salts. Their alcoholic solution shows a yellow-red fluorescence. Phenosafranine is not very stable in the free state; its chloride forms green plates. It can be readily diazotized, and the diazonium salt when boiled with alcohol yields aposafranine or benzene induline, C18H12N3. F. Kehrmann showed that aposafranine could be diazotized in the presence of cold concentrated sulfuric acid, and the diazonium salt on boiling with alcohol yielded phenylphenazonium salts. Aposafranone, C18H12N2O, is formed by heating aposafranine with concentrated hydrochloric acid. These three compounds are perhaps to be represented as ortho- or as para-quinones. The "safranine" of commerce is a ortho-tolusafranine.

A counterstain is a stain with color contrasting to the principal stain, making the stained structure more easily visible. An example is the malachite green counterstain to the fuchsine stain in the Gimenez staining technique. Another example is eosin counterstain to haematoxylin in the H&E stain. Also in Gram staining, crystal violet stains only Gram-positive bacteria, and safranin counterstain is applied which stains all cells, even allowing the identification of Gram-negative bacteria as well.


d) In this Gram test, we need to use young bacteria cultures because we need to differentiate between Gram-positive bacteria (large Peptidoglycan layer on outer surface of cell) and Gram-negative bacteria.

e) Gram staining is an empirical method of differentiating bacterial species into two large groups based on the chemical and physical properties of their cell walls.

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan which is capable of retaining the violet dye/iodine complex. Gram-negative bacteria have a thin cell wall made of a layer of peptidoglycan. In addition to an inner membrane, they also have an outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.

The decolorizing mixture causes dehydration of the multilayered peptidoglycan in the Gram-positive cell wall, thus decreasing the space between the molecules and causing the cell wall to trap the crystal violet-iodine complex within the cell. But in Gram-negative bacteria, the decolorizing mixture acts as a lipid solvent and dissolves the outer membrane of the Gram-negative cell wall. The thin layer of peptidoglycan is unable to retain the crystal violet-iodine complex and the Gram-negative cell is decolorized. The decolorization step is the crucial one, and requires some degree of skill, as Gram-positivity is not an all-or-none phenomenon.

As a rule of thumb, Gram-negative bacteria are more dangerous as disease organisms, because their outer membrane is often hidden by a capsule or slime layer. Additionally, Gram-negative bacteria have lipopolysaccharide in their outer membrane. Lipopolysaccharide is an endotoxin which increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Gram-positive infections are generally less severe because the human body does not contain peptidoglycan, and in fact the human body produces an enzyme called lysozyme which attacks the open peptidoglycan layer of Gram-positive bacteria. Gram-positive bacteria are also much more susceptible to beta-lactam antibiotics, such as penicillin.


CONCLUSION;-

Gram staining (or Gram's method) is an empirical method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. While Gram staining is a valuable diagnostic tool in both clinical and research settings, not all bacteria can be definitively classified by this technique, thus forming Gram variable and Gram indeterminant groups as well.

Gram staining is used to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell walls. Gram staining is not used to classify archaea, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.


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