TECHNIQUES IN MICROBIOLOGY I

EXPERIMENT 4.1: INOCULATING OF BACTERIA IN YOGURT. EXPERIMENT 4.2: VIABLE COUNT OF BACTERIA IN YOUGURT. EXPERIMENT 4.3: STAINING OF BACTERIA.

Name Group Lectures name

: Mohd Izwan B. Ibrahim : ASD5Gn : Sir Azhar B. Zulkifli

EXPERIMENT 4.1 Inoculating of bacteria in yogurt.

OBJECTIVES 1. To inoculate bacteria from yogurt by using streaking technique. 2. To determine the presence of bacteria in petri plates. MATERIALS Sample. 1. Fresh yogurt. Apparatus. 1. Inoculating loop. 2. Bunsen burner. 3. Indelible marker. 4. Incubator. Chemicals. 1. 2 sterile nutrient agar plates.

METHODS. 1. Inoculating loop was placed in the Bunsen burner flame until the loop is red hot. 2. The loop was allowed to cool and dipped into the yogurt. 3. The lid of the sterile agar plate was slightly lifted and the contents of the inoculating loop were spread over the surface of the agar by using zig-zag motion. 4. The lid of the plate was closed and the loop was returned to the Bunsen burner until red hot. 5. The base of the plate was labelled with an indelible marker. 6. The procedures were repeated for the second plate. 7. The two halves were tape together. The plates then were placed in the incubator upside down. 8. The temperature of the incubator was set at 35oC for three days. 9. The plates were observed every day and the appearance of the colonies were recorded in Table 4.1

EXPERIMENT 4.2 Viable Count of Bacteria in Yogurt.

OBJECTIVES The main objectives of this experiment are: i. ii. iii. To make a serial dilution of yogurt sample. To transfer the bacteria of petri plates. To compare the number of bacteria presents in Petri plates.

MATERIALS Sample 1. Any yogurt samples. Apparatus. 1. 1 mL micropipettes. 2. 200 L micropipettes. 3. 10 mL graduated pipettes. 4. Test tubes and test tube rack. 5. Cotton wool. 6. Oven indelible marker. 7. Bunsen burner. 8. Aluminium foil. 9. Glass spreader. 10. 1mL and rack. 11. 200 L tips 12. Rack. Chemicals 1. Sterile nutrient agar plates. 2. 100mL 0.9% v/v saline solution. 3. 70% v/v alcohol.

METHODS Sterilization of Apparatus. 1. Cotton wool was plugs in each six test tubes and cover plugs loosely with aluminium foil. 2. Place a small piece of cotton wool on each of eight 1mL graduated pipettes and the 10 graduated pipette and wrap each pipette separately in aluminium foil. 3. The test tube and pipettes was place in a hot oven at 160oC for 60 minutes. 4. Allow all apparatus to cool before use.

Serial Dilution of Yogurt and spreading on Agar Plate. 1. The six sterile plugged test tubes were labelled as Y1 and Y6 and the aluminium foil covers from the plug was removed. 2. The three sterile nutrient agar plates were labelled with P1, P2 and P3 each. 3. 9 mL of sterile distilled water was transferred to each of the six test tubes using the techniques below : a) The bottle cap containing the sterile solution saline solution was removed. b) 9 mL of saline solution was drew up using the sterile solution 10 mL graduated pipettes. c) The bottle cap was replaced. d) The plug from the first test tube Y1 was removed. e) 9 mL of saline solution was transfer to the test tube. f) The test tube plug was replaced. g) The process for the five remaining test tubes was repeated. 4. The fresh yogurt sample was shaked and 1 mL of this yogurt was transfer using a sterile 1 mL micropipette to Y1, removing and replacing the plug before. This will give 10X dilution. 5. The Y1 test tube was gently shaked to ensure thorough mixing. 6. 1 mL from tube Y1 transfer to the next tube Y2 by using a fresh tip, the step 5 and step 6 was repeated until the last test tube, Y6. 7. 100 L is transferred from the test tube Y4 using 200L micropipettes to the sterile plate labelled P1 (X10 ), lifting the lid by a minimal amount.

8. The glass spreader was dipped in 70% alcohol to allow alcohol excess to drip off and hold the spreader vertically in Bunsen burner flame. 9. The spreader was cooled and spread the diluted yogurt sample over the surface of the plate. 10. The spreader was re-sterile. 11. The same micropipette and fresh tips was using, 100 L is transferred from the test tube Y5 to the sterile plate labelled P2 (X10 ), lifting the lid by a minimal amount. Step 8 to 10 is repeated. 12. The step 11 was repeated for the test tube Y6 to sterile plate labelled P3 (X10 ). spread. 14. The six plates upside down at 35 C for about three day in incubate. 15. The plates were examined for bacterial growth and count the number of individual colonies on each plate. The results were recorded in Table 4.2 and use them to calculate the number of bacteria in 1 mL of undiluted yogurt. 16. The colony counter was used to estimate the total number of bacteria on the plate. 13. The lid of the plates should then be taped down to avoid the risk of pathogens being

EXPERIMENT 4.3 Staining of Bacteria.

OBJECTIVES. The main objectives of this experiment are: I. II. To stain bacteria for examination under a light microscope. To identify the morphology of bacteria.

MATERIALS Sample. 1. Bacteria cultured from previous experiment. Apparatus. 1. Wire loop. 2. Bunsen burner. 3. Glass slides. 4. Forceps. 5. Staining rack ( set up over sink or dish ). 6. Distilled water in wash bottle. 7. Blotting paper. 8. Immersion oil. 9. Compound light microscope. 10. Oil immersion lens.

Chemicals 1. Basic stain crystal violet ( 0.5 % w/v aqueous ). 2. Mordant Lugols iodine. 3. Decoloriser acetone alcohol ( 50:50 acetone : absolute alcohol ) 4. Counter stain safranin ( 1% w/v aqueous ).

METHODS. Preparing a Smear of Bacteria.

1. The wire loop was put onto the fire and cooled. 2. A loop-full or two of tap water was placed on the center of a clean slide. 3. The selected bacterial colony from Experiment 4.1 was taken by touching with the wire loop lightly. (The lid was opened slightly for safety reason). 4. The bacteria was spreaded over the slide and mixed with the water gently. 5. The bacteria was spreaded over the slide by using the loop to cover an area 3x1 cm. It is important to achieve the correct thickness of the smear. 6. The loop was put onto the fire again. 7. The smear was allowed to dry in air for a few minutes. 8. The bacteria was fixed. The slide was carefully holded with the forceps and passed horizontally just over a yellow Bunsen burner flame for three times. (Do not overheat). The cytoplasm was coagulated and sticked to the slide by fixing the bacteria.

Prepare a Staining of Bacteria 1. This should be done on a rack over a sink or dish. Note: A rack can be made by arranging two glass rods or metal rods across the sink or dish 5 cm apart and absolutely horizontal and can be supported by plasticine. 2. Put on prepared glass slide (from step A) on a rack to stain the bacteria. 3. Flood the slide with crystal violets stain. Leave for 30 seconds. This makes all bacteria violet. 4. Then, flood with Lugols iodine and leave for 30 seconds. Wash off the iodine with distilled water. The iodine fixes the stain more permanently into the cells. 5. Flood the slide with acetone-alcohol until no more colour is seen to come off (about 3 seconds). Immediately wash with water to prevent excessive decolourisation. Repeat if necessary. This decolorizes Gram-negative bacteria, and Gram-positive bacteria stay violet. 6. Flood the slide with Safranin and leave for 1 minute. Wash off the slide with distilled water. Gently dry the slide between sheets of clean blotting paper and allow drying in air. (Safranin is described as a counterstain. It is used after the crystal violet to stain any Gram-negative bacteria red).

7. Apply a drop of immersion oil and examine under the oil immersion lens (fresh mount). Figure 4.3 show an example of bacterial smear on a slide as viewed at 1000X magnification. 8. Sketch your result in Table 4.3 and give the characteristics of bacteria. 9. In order to make a permanent preparation, place a drop of mounting on the slide, cover with a coverslip and press down slowly and carefully to exclude air.

Discussion. It is a differential staining 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. This reaction divides the eubacteria into two fundamental groups according to their stainability and is one of the basic foundations on which bacterial identification is built. The primary stain renders all the bacteria uniformly violet. After a minute of exposure to the staining solution, the slide is washed in water. The smear is treated with few drop of Gram's Iodine and allowed to act for a minute. This results in formation of a dye-iodine complex in the cytoplasm. Gram's iodine serves as a mordant. The slide is again washed in water and then decolorized in absolute ethyl alcohol or acetone. A mixture of acetone-ethyl alcohol (1:1) can also be used for decolourization. The process of decolourization is fairly quick and should not exceed 30 seconds for thin smears. Acetone is a potent decolourizer and when used alone can decolorize the smear in 2-3 seconds. A mixture of ethanol and acetone acts more slowly than pure acetone. Decolourization is the most crucial part of Gram staining and errors can occur here. Prolonged decolourization can lead to over-decolorized smear and a very short decolourization period may lead to under-decolorized smear. After the smear is decolorized, it is washed in water without any delay. The smear is finally treated with few drops of counterstain such as dilute carbol fuchsin, neutral red or safranin.

Before start inoculation process the site of the experiment must spray with alcohol (acetone) to kill the bacteria on that table. Then, wire loop must be sterilised by fire the wire loop until red. Then let it cool for a while, then inoculation process can be proceed. To repeat the inoculation, the wire loop need to fire again with fire. Besides, the process of inoculation must undergoes near to fire source to avoid contaminant. The heat of the fire can kill the bacteria.

In the grams staining method, we should follow the staining method correctly. For examples we need to cover the specimen with crystal violet for 30 seconds. Then follow with Lugols iodine for another 30 seconds. Then we need to wash with acetone for 3 seconds. If we washed the specimen with acetone more than 3 seconds the cell could be bleach or vanish. After wash the specimen with acetone for 3 seconds, quickly wash the specimen with distilled water until all the acetone is clear. Then we need to cover the specimen with safranin (counter stain) for 1 minute. This steps is to ensure the tissue is stained completely. Then wash the specimen with distilled water.

The bacteria present in the sample is Lactobacillus sp.. Gabriela Perdigon proposed that yoghurt is a product obtained by the fermentation of milk with cultures of Streptococcus thermophilus and Lactobacillus bulgaricus.

Conclusion. From the result we got, our petri dish was contaminant. This is due to lack of precaution steps we took when handling the experiments. The bacteria we observed are negative gram bacteria. This can be explained form the colour of the staining is red and little bit pink.

References. 1. www.microrao.com. 2. Cambell Biology 9th edition.

TECHNIQUES IN HISTOLOGY

EXPERIMENT 3.1 : PREPARING A SLIDE OF PLANT TISSUES EXPERIMENT 3.2 : PREPARING A SLIDE OF INSECT TISSUES

Name Group Lectures name

: Mohd Izwan B. Ibrahim : ASD5Gn : Sir Azhar B. Zulkifli

EXPERIMENT 3.1 Preparing a Slide of Plant Tissues.

OBJECTIVES. i. ii. iii. To make a thin slice by using hand-cutting method To prepare, stain and make a slide of plant sample To illustrate the observation of plant sample.

MATERIALS. Samples. 1. A young dicot (balsam, Impatiens balsamina)

Apparatus. 1. Compound microscope, slide and coverslip.

Chemical. 1. Acetic-eosin.

METHODS. 1. 2 cm of a young root of balsam was obtained. It was placed on a microscope slide or plate. 2. The root was cut very thin slices (transverse and cross section) through the region of maturation. 3. The thinnest section was selected and transferred it in to a drop of water on another slide. The cover slip was placed over a specimen. 4. The slide was observed using compound microscope. The observation was sketched and labelled in the space provided in Table 3.1. 5. The tissue systems in young root was identify; vascular cylinder (phloem, xylem), cortex and epidermis. 6. The steps 1 until 4 were repeated by using stem of the dicot. 7. In order to have a permanent slide, the coverslip was removed with a jet water, then air dry and mounts in Canada balsam or similar media.

EXPERIMENT 3.2 Preparing a Slide of Insect Tissues.

OBJECTIVES. The main objectives of this experiment are: i. ii. iii. To make a slide by using squash technique. To prepare, stain and make a slide of insect sample. To illustrate the observation of insect sample.

MATERIALS Sample. 1. A male locust (Example: locust, Valanga nigricornis) Apparatus. 1. Dissecting and compound microscope. 2. Slide and coverslip. 3. Small dissecting dish. 4. Pin. 5. Glass rod. 6. Hotplate or Bunsen burner. 7. Filter paper. Chemical. 1. Acetic-orcein. 2. Canada balsam. 3. Dry ice .

METHODS. 1. Pin out a freshly killed male locust on a small dissecting dish with dorsal side uppermost. Pin out the wings on each side. 2. Open up the abdomen by a mid-dorsal longitudinal cut. Pin back the body wall. 3. Using a dissecting microscope, identify the testis. Together with fat, these make up an oval body lying above the gut in abdominal segment (segments 5 and 6). Transfer the testes on a microscope slide. 4. Remove as much of the fat (yellow) as you can, leaving only the white tubules of the testis. Two or three tubules are sufficient. 5. Add several drops of acetic-orcein stain and put on a coverslip. 6. Cover with filter paper and squash gently. This helps to spread the chromosomes. 7. Warm up the slide on a hotplate or Bunsen burner for about 10 seconds to intensify the staining. 8. Observe your slide using microscope, and sketch your specimen. Take photo if you have digital microscope. Examine the stages in meiosis. Draw and label the chromosomes in Table 3.2. 9. In order to have a permanent slide, remove the coverslip by freezing in dry ice or liquid nitrogen and the slide must be throughly air dry. 10. Wash the slide with a jet of water to remove the excess dye, then air dry the material once again and mount in Canada balsam or similar media.

DISCUSSION. Water-carrying cells are also known as Xylem while food-carrying cells known as Phloem. Both xylem and phloem are vascular tissues found in a plant. Xylem is a tubular structure which is responsible for water transport from the roots towards all of the parts of the plant. Phloem is also a tubular structure but is responsible for the transportation of food and other nutrients needed by plant. Xylem imports water and minerals while Phloem transports water and food. Xylem exists as non-living tissue at maturity, but phloem is living cells. Xylem are hard wall cells transport water and mineral nutrients while Phloem are relatively soft -walled cells transport organic nutrients. "Hardness and softness" is a function of the amount of lignification and extractive content of the individual cell walls not there location in the tree.

The characteristics of water-carrying cells (xylem) and food-carrying cells (phloem) also can be compared as shown below: Xylem Phloem

1. It is a dead complex permanent tissue. 1. It is a living complex permanent tissue. Sapwood is xylem and mostly alive. Inner phloem is alive. Outer phloem is dead. 2. It is the principal water conducting tissue 2. It is the principal food conducting tissue of vascular plants. found in vascular plants. Actually only the first .2-.7mm of the phloem is functional in food transport. The rest is non3. Xylem consists of tracheids,vessels, xylem parenchyma and xylem fibers. Also ray parenchyma. Also the xylem of softwoods (Gymnospermae) do not contain vessels. functioning (ref. Esau's Plant Anatomy, 3rd ed. 2006, Chap.14) also (ALFIERI, F. J., and R. F. EVERT. 1968. Seasonal development of the secondary phloem in (Pinus. Am. J. Bot. 55, 518-528.) 3. It consists of sieve cells, sieve tube elements, phloem parenchyma and phloem fibres. There are also phloem ray cells.

Xylem consists of the inner heartwood (the dead part) and the outer portion called the sapwood (the mostly living part.) Xylem "wood" from the Greek xylon. The actual transport of the water and minerals from the roots (sap) is carried out by the outer portion of the sapwood (nearest the bark).The largest percentage of the sap is transported in the first few growth rings.

During the slide-making process, there are several precautionary steps that need to be engaged. First of all, the slide needs to be check whether it is clean or not. If not, the slide is supposed to be washed by soap-water, then, wipe it with ethyl alcohol (ethanol). This procedure will make the slide clean and sterilized. When staining the slide, do not stain the whole surface of slide because it will cause the slide to be messy. Just stain the area that need to be examine.

To place the coverslip, the slip must be place properly and air bubbles must be avoided to get the best results. While observing the slide, always prefer to observe under 10X first. This will give you an idea of the location of a good area for observation. After this you may prefer to switch over to 45X. To observe 100X objective in compound microscopes, always used as an oil-immersion objective, so do not ever observe at a specimen at 100X without oil. I observed that many students are tempted to observe the 100X objective without using oil. This will eventually break the lens of 100X objective. Staining is an auxiliary technique used in microscopy to enhance contrast the microscopic images. Stains and dyes are frequently used in biology and medicine to highlight structure in biology tissues for viewing, often with the aid of different microscopes. Stain may be used to define and examine bulk tissues, for an example muscle fiber or connective tissue, cell populations (classifying different blood cells for instance) or organelles within individual cells. In biochemistry it involves adding a class specific (DNA, proteins, lipids, carbohydrates) dye to a substrates to qualify or quantify the presence of a specific compound. Staining and fluorescent tagging can serve similar purposes biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acid in gel electrophoresis. Staining is not limited to biological material, it can also be used to study the morphology of other materials for example the lamellar structure of semi-crystalline polymer or the domain structures of block copolymers.

The chemical reactions involved is Eosin. Eosin is the most often used as a counterstain to haematoxylin, imparting a pink or red colour to cytoplasmic material, cell membranes, and some extracellular structures. It also imparts a strong red colour to red blood cells. Eosin may also be used as a counterstain in some variants of Gram staining, and in many other protocols. There are actually two very closely related compounds commonly reffered to as eosin. Most often used is eosin Y (also known as eosin ws of eosin yellowish) it has a cery slightly yellowish cast. The other eosin compound is eosin B (eosin bluish or imperial red) it has very faint bluish cast. The two dyes are interchangeable, and the use of one or the other is more a matter of preference and tradition. The second is acetic-orcein that is needed in stain of chromosome of plants or animals. In this experiment, we used aceticorcein to stained the chromosomes in testis of locust.

CONCLUSION. For the conclusion of this experiment, we can make a thin slice by using hand-cutting method and make a slide by using a squash method. We also can prepare, stain and make a slide of plant and insect sample. Lastly, we also can illustrate the observation of plant and insect sample.

REFERENCES. CAMPBELL BIOLOGY, NINTH EDITION. Biology Techniques and Skills (Laboratory Manual)