EXCERSICE 3C: MICROSCOPIC CHARACTERISTICS

Group #4 Crystal Jade Mendoza Justine Gonzales Millare Steffi Muriel Oslyn Opilas Em Osias Ojochenemi Peters Tanja Philipp Jamie Pooten INTRODUCTION: Microscopic examination of various bacteria The type of cell wall that a bacterium has can be determined by utilizing various staining techniques. One such technique is called a Gram stain. This technique involves a series of stains (crystal violet, Gram iodine and safranin) that are applied to bacterial cells on a slide. Gram positive bacteria have a thicker peptidoglycan layer, thus these bacteria are able to retain the crystal violet/iodine color and appear purple. Gram-negative bacteria have less peptidoglycan than gram-positive bacteria. As a result, gram-negative bacteria will lose the purple color but retain the safranin and appear pink/red instead. This is due to their cell wall having less peptidoglycan and being more complex with various proteins, polysaccharides and lipids. The cell wall affords some protection to bacteria. In addition to the cell wall, many bacteria can secrete a gelatinous capsule around themselves. The capsule may allows the bacteria to adhere to substances, but may also decrease the host cells ability to destroy them. The lipopolysaccharide layer of gram-negative bacteria also increases protection by decreasing the ability of the host to rid itself of these bacteria. This outermost layer may even be toxic. Of greater importance is this layers ability to make it resistant to antibiotics by preventing drug entry into the organism.

OBJECTIVE: In this Activity, you will do the different staining method to the different bacteria given and examine them under microscope. Under the microscope, you could see the shape, arrangement, color of the unknown bacteria which will help you classify what bacteria or what kind of bacteria you are looking at. In this section, you will examine several fixed slides of bacteria, some of which cause human diseases of great importance.

THE DIFFERENT STAINING METHOD

(DISCUSSION)

Direct simple Staining: Simple stains use just only one dye, and highlight cell morphology.
Procedure
1. Clean and dry microscope slides thoroughly. 2. Flame the surface in which the smear is to be spread. 3. Flame the inoculating loop. 4. Transfer a loop full of tap water to the flamed slide surface. 5. Reflame the loop making sure the entire length of the wire that will enter the tube has been heated to redness 6. Remove the tube cap with the fingers of the hand holding the loop. 7. Flame the tube mouth. 8. Touch the inoculating loop to the inside of the tube to make sure it is not so hot that it will distort the bacterial cells; then pick up a pinhead size sample of the bacterial growth without digging into the agar. 9. Reflame the tube mouth, replace the can, and put the tube back in the holder. 10. Disperse the bacteria on the loop in the drop of water on the slide and spread the drop over an area the size of a dime. It should be a thin, even smear. 11. Reflame the inoculating loop to redness including the entire length that entered the tube. 12. Allow the smear to dry thoroughly. 13. Heat-fix the smear cautiously by passing the underside of the slide through the burner flame two or three times. Test the temperature of the slide after each pass against the back of the hand. It has been heated sufficiently when it feels hot but can still be held against the skin for several seconds. Overheating will distort the cells. 14. Stain the smear by flooding it with one of the staining solutions and allowing it to remain covered with the stain for the time designated below. Methylene blue - 1 minute // During the staining the slide may be placed on the rack or held in the fingers. 15. At the end of the designated time rinse off the excess stain with gently running tap water. Rinse thoroughly. Air dry.

Purpose: To recognize the three basic shapes of bacterial cells. Principle: In order to observe most bacterial cells using bright field microscopy the cells must be dark
enough to see, that is they must have contrast to the light. To create contrast a simple stain can be used.

Simple stains use basic dyes (Methylene blue) which are positively charged. These positive dyes interact with the slightly negatively charged bacterial cell wall thus lending the color of the dye to the cell wall.

Negative Staining: In negative staining, the microbes repels the dye and it stains the
background. Dyes may be used alone and in combination.

Procedure:
1. Place a very small drop (more than a loop full--less than a free falling drop from the dropper) of nigrosin near one end of a well-cleaned and flamed slide. 2. Remove a small amount of the culture from the slant with an inoculating loop and disperse it in the drop of stain without spreading the drop. 3. Use another clean slide to spread the drop of stain containing the organism using the following technique. 4. Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean slide toward the drop forming an acute angle and draw that slide toward the drop until it touches the drop and causes it to spread along the edge of the spreader slide. Maintaining a small acute angle between the slides, push the spreader slide toward the clean end of the slide being stained dragging the drop behind the spreader slide and producing a broad, even, thin smear.

5. Allow the smear to dry without heating. 6. Focus a thin area under oil immersion and observe the unstained cells surrounded by the gray stain.

Purpose:
fixing.

To determine morphology, arrangement and size of bacteria, that may be affected by heat

Principle: The negative stain uses the dye nigrosin, which is an acidic dye.

By giving up a proton (as an acid) the chromophore of the dye becomes negatively charged. Because the cell wall is also negatively charged only the background around the cells will become stained, leaving the cells unstained

Gram Staining: the most universal siagnostic staining technique for bacteria.
Procedure
A. Slant Cultures 1. Prepare and heat-fix smears. 2. Prepare the smears of S. epidermidis and N. sicca on a second slide. Heat-fix. 3. Stain the slides as follows: a. Flood the crystal violet for one minute. b. Pour off excess dye and wash gently in tap water (5secs) and drain the slide against a paper towel. c. Expose the smears to Gram's iodine for one minute by washing with iodine, then adding more iodine and leaving it on the smear until the minute is over. d. Wash with tap water and drain carefully. (Do not blot.) e. Wash with 70% ethanol for 30 seconds. f. Wash with tap water at the end of the 30 seconds to stop the decolorization. Drain. g. Counterstain with safranin for 30 seconds. h. Wash, drain, blot, and examine under oil. B. Broth Cultures 1. Because the smear made from the broth will be a thin smear and nearly invisible to the naked eye even after staining, it may be advisable to draw a ring with a felt open on the under side of the slide to mark the area in which the broth smear will be made. Also, when making a smear from broth do not add a drop of water to the slide. 2. Hear-fix the smears, Gram stain them with the above procedure, and examine them. When focusing the broth smear use the technique suggested for thin smears. 3. Compare the appearance of the cells in the two smears.

Purpose: To differentiate between Gram-positive and Gram-negative organisms, while simultaneously

learning about the cellular morphology and arrangement.

Principle: Gram-positive cells have a thick peptidoglycan cell wall that is able to retain the crystal
violet-iodine complex that occurs during staining, while Gram-negative cells have only a thin layer of peptidoglycan. Thus Gram-positive cells do not decolorize with ethanol, and Gram-negative cells do decolorize. This allows the Gram-negative cells to accept the counter stain safranin. Gram-positive cells will appear blue to purple, while Gram-negative cells will appear pink to red.

Acid fast Staining: an imposrtant duiagnostic stain that differentiates acid fast bacteria (pink) from
nonacid fast bacteria (blue).

Procedure
1. Prepare smears of organisms to be stained. 2. Heat fix the smears. 3. Cut or tear absorbent paper (bibulous paper) to fit the slide leaving one end for handling. Do not allow the paper to protrude beyond the slide, but the smears must be covered. 4. Place the slide on steamer. 5. Saturate the paper with carbolfuschin. 6. Heat the slides with a steamer until steam can be seen rising from the surface, maintain steaming for 5 minutes. As the paper begins to dry during the staining process add a drop or two of carbolfuschin to keep the slide moist. Adding too much stain will cool the slide (and drip on the bench). Overheating the slide or letting it dry will distort the cells. Under heating the slide will fail to stain acid-fast cells. 7. At the end of staining remove the paper with tweezers and wash the slide thoroughly. 8. Drain the slide. 9. Decolorize with acid-alcohol for 30 seconds. 10. Rinse, drain, and counterstain with methylene blue for 45 seconds. 11. Rinse, blot, and examine. First observe each organism on its separate smear. Then examine the mixed smear. 12. Acid-fast organisms will appear red and non-acid-fast organisms will be blue.

Purpose: To differentiate between acid-fast and non acid-fast bacteria. Principle: Some bacteria contain a waxy lipid, mycolic acid, in there cell wall. This lipid makes the
cells more durable and is commonly associated with pathogens. Acid fast cell walls are so durable that the stain (carbol fuschin) must be driven into the cells with heat. The cells are then decolorized with acidalcohol, all other cells will decolorize with this strong solvent, but acid fast bacteria will not. Other cells are then counterstained with methylene blue.

Spore Staining
Procedure
1. Prepare smears of organisms to be tested for endospores. 2. Heat fix the smears. 3. Cover the smears with a piece of absorbent paper cut to fit the slide and place the slide on a wire gauze on a ring stand. 4. Saturate the paper with malachite green put it in steamer, heat the slide until steam can be seen rising from the surface. Remove the heat and reheat the slide as needed to keep the slide steaming for about three minutes. As the paper begins to dry add a drop or two of malachite green to keep it moist, but don't add so much at one time that the temperature is appreciably reduced. DO NOT OVERHEAT. The process is steaming and not baking. 5. Remove the paper with tweezers and rinse the slide thoroughly with tap water. 6. Drain the slide and counterstain 45 seconds with 0.5% safranin. 7. Wash, blot, and examine. 8. The vegetative cells will appear red and the spores will appear green.

Purpose: To differentiate between organisms that can produce endospores and those that cannot. Also
allows differentiation based on the position of the endospore in the cell

Principle: Spores have a durable outer coating that is composed of the protein keratin. This keratin coat
resists staining so in order to stain a spore the primary stain, malchite green, must be heated to drive the stain into the spores. Vegatative cells are then decolorized with water and 0.5% safranin is used to counterstain. Thus endospores are stained green, while vegetative cells are stained red.

Flagella Staining:
Procedure:
A. Bacterial Suspension 1. From an agar slant culture: a. Suspend a loopful of bacteria in 2 ml distilled water to obtain an opalescent suspension. b. Allow the suspension to stand undisturbed for 15 to 20 minutes while flagella are regenerated and extended. B. Slides 1. Select new or unscratched slides. 2. Clean slides with cleaning powder as directed. 3. Flame one of the slides and allow it to cool. 4. Make a heavy wax line on the flamed side along the margin of one end, along both sides to within about one inch of the other end, and across the slide to complete the rectangle. Handle the slide by the unmarked end only. The wax line creates a retaining wall to allow a pool of stain to surround the cells during the 15 to 15 minute staining period. C. Preparation of smear 1. Handling the suspension carefully remove a large loopful of the suspension and place it at one end of the rectangular area. 2. Tilt the slide to permit the suspension to run down to the other end of the slide. If the drop fails to run, add another drop. 3. Air dry the film. Do not heat. 4. Place the slide on a horizontal staining rack. D. Staining procedure 1. Add about 1 ml. of the Flagella Stain solution (one dropper full) to the smear and allow to stain for 1015 minutes. The solution should not be allowed to flow outside the waxed area. 2. Flood off the stain by adding tap water to the slide while it remains on the rack. Do not tip the slide before this is done. 3. Drain and flood the slide with carbol fuchsin for one minute. Rinse by flooding. Drain and air dry. Do not blot. E. Examination of slide 1. With the naked eye identify the line made by the drop as it ran down the slide. 2. Position the slide so that the edge of the run is under the objective. 3. Focus the edge of the run under low power, as oil, and examine it using the oil immersion objective. 4. Identify cells as distinguished from dye and debris which will adhere to the slide because of the mordant. Cells will be small rods and have regular outlines. They will be more plentiful near the lower end of the run and may look larger than usual because of the mordant. 5. Once cells are identified follow along the edge of the run, examining cells until some are found with flagella attached. Flagella are much longer than the cell and often look like faint hairs. 6. Ideally, cells should be located which are isolated enough to determine unequivocally the arrangement

of the flagella and, for the Pseudomonas, the number of flagella per cell. 7. Draw the cells and their flagella.

Purpose: To determine the presence/absence and location of flagella on various microorganisms Principle: Because bacterial flagella are very thin and fragile a special stain (flagella stain) is prepared
that contains a mordant. This mordant allows piling of the stain on the flagella, increasing the thickness until they become visible. Various arrangements of flagella are seen on different cells.

-------------------------------------Capsule staining:
Procedure:
1. Place a single drop of India ink on a clean microscope slide, adjacent to the frosted adge. 2. Using a flamed loop and sterile technique, remove some K. pneumoniae (or the organism you want to stain) from your tube or plate and mix it into the drop of India ink. Be sure there are no large clumps of organism, but try to avoid spreading the drop. 3. Place the end of another clean microscope slide at an angle to the end of the slide containing the organism. Spread out the drop out into a film. This is done by contacting the drop of India ink with the clean microscope slide and using the capillary action of the dye/ slide to spread the India ink across the smear. Refer to the Negative Stain portion of this handout for a diagram. 4. Allow the film to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule! 5. Saturate the slide with crystal violet for 1 minute. 6. Rinse the slide gently with water. 7. Allow the slide to air dry. DO NOT heat or blot dry!!!! Heat will melt the capsule! 8. Observe the slide under the microscope, using proper microscope technique.

Purpose: The capsule stain is a differential stain which selectively stains external capsules
surrounding bacterial cells. Principle: Capsules are formed by organisms such as Klebsiella pneumoniae . Most capsules are composed of polysaccharides, but some are composed of polypeptides. The capsule differs from the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer outside the cell wall. Some capsules have well-defined boundaries, and some have fuzzy, trailing edges. Capsules protect bacteria from the phagocytic action of leukocytes and allow pathogens to invade the body. If a pathogen loses its ability to form capsules, it can become avirulent.

Importance of Fixing/Preparing a Bacterial Smear for Staining

Before staining and observing a microbe under a microscope, a smear must be prepared. The goal of smear preparation is to place an appropriate concentration of cells on a slide and then cement them there so that they do not wash off during the subsequent staining procedure. The best smears are made from bacteria that have grown on a solid surface such as an agar slant or plate. A bit of growth from a culture is mixed with distilled or tap water to form a slightly turbid solution and this is spread on a clean grease free slide. When staining broth cultures, a drop of broth is transferred directly to a slide, using no extra water. The procedure for making a smear is as follows: 1. If more than one culture is to be examined using the same stain, it is possible to prepare up to 6 smears on the same slide. Before preparing the slide, divide it into the appropriate number of sections and clearly label each section on the underside of the slide. 2. If your culture has been grown on a agar slant or agar plate. Place a small drop of water on a clean, grease-free slide. Next, using a sterile loop or straight wire needle, transfer a bit of the growth to the drop of water and rub the needle around until the material is evenly emulsified. Spread the drop over a portion of the slide to make a thin film. The suspension should be only slightly turbid. 3. If you are using a broth culture, the broth culture must have clearly visible turbidity. Transfer a loopful of culture from the broth onto a clean grease free slide. Spread the drop over a portion of the slide to make a thin film. 4. Allow the film to air-dry. To get a good stain, it is important to let the smear dry completely. Excess water left on the slide will boil during the fixing stage, causing most microbe present to rupture. Rushing this step will result in a poor final stain. 5. Once dry, "fix" the smear to the slide by passing the bottom of the slide through the tip of the burner flame several times for a one second. After heat fixing, touch the heated portion of the slide to your hand. It should be comfortably warm, but not burning hot. 6. Take care not to under-fix (the smear will wash off) or over-heat (the cells will be ruptured or distorted) the slide. The correct amount of heat fixing is learned by experience. Allow the smear to cool and apply the stain.

CONCLUSION: It is important to examine bacteria / microbes, to know which can benefit us or harm us. Staining techniques is important in examining bacteria to allow better viewing and shows more details.

FW-A11
Bacteria Indentity: Staphylococcus ?? DIRECT: simple staining Shape: Coccus; Grape-like Arrangement: Clusters Color: Violet Spore: Negative Because It is color Red

Gram Staining: the bacterium (FW-A11) stained dark violet which indicates that: Bacteria FW-A11 is gram positive bacterium. It does have a thick peptidoglycan cell wall and therefore retains the crystal violet stain. Acid fast stain: Negative It is blue The carbolfuchsin washes out and you see the methylene blue

FW-A10
Bacteria Identity: Escheria coli Direct: simple staining
The Bacterium FW-A10 is gram negative, it has thin peptidoglycan cell wall and stains a pink colour on a gram stain from the counterstain safranin

Shape: Rod shaped; bacilli; diplobacilli Arrangement: Clusters Color: Blue

Spore Staining:
Non-sporulating because bacteria FW-A10 is a gram negative bateria

Gram Staining: Acid Fast Staining: Negative Non Acid Fast (blue)

FW-A18
Bacterial Identity: ??? Direct: simple staining Shape: Rod shaped (coccubacillus) Arrangement: singles, clusters // not sure Color:

Gram Staining: The Bacterium FW-A18 is a gram

negative bacterium

Spore Staining: Non Sporulating (No spores)

Acid Fast Staining: Cant Identify