Preparing and Gram-staining a bacteriological smear

Transcription

1 College of Life Sciences and Technology Medical Laboratory Science (Applied Learning) AP (52) Module 4: Practical Preparing and Gram-staining a bacteriological smear Date Time Class Venue October 22, 2016 (Sat) 10:00-17:30 A and B HKU L19 Practical Supervision/Demonstrators To be announced Technical Assistance To be announced References Estridge BH and Reynolds AP (2008) Preparing and Gram-staining a bacteriological smear. In Basic clinical laboratory techniques, pp USA: Delmar Ridley JW (2011) Microbiology. In Essentials of clinical laboratory science, pp USA: Delmar Turgeon ML (2012) Introduction to Microbiology. In Linne and Ringsrud's clinical laboratory science: the basics and routine techniques, pp USA: Mosby 1

2 Objectives 1. To demonstrate the proper procedures for preparing a bacterial smear, and for the microscopic examination of Gram-stained smear. 2. To provide a working experience to the students in using the instruments for routine preparing and Gram-staining a bacterial smear. 3. To understand the principle of the Gram stain reaction, and correlate its importance to the bacterial identification in contemporary medical microbiology. Background To identify the bacteria that is the etiologic or causative agent of an infection correctly, the microbiologists should carry out the microscopic observations (smear preparation and staining), bacterial cultivation and biochemical tests. Direct examination via microscopic observation of bacterial smears helps to determine the purity of a culture, to select the type of medium for culturing. For instance, tentative diagnosis can be made from the direct examination of bacterial smears from the clinical specimens. Bacteria are small and have little natural color for most species, they are difficult to be observed microscopically unless they are stained. Therefore various staining methods has been devised to enable the microbiologists to examine bacteria. Gram stain is the most frequently performed and is an important differential stain used in microbiology laboratory. It helps to differentiate various types of bacteria that have similar morphologic features. The Gram stain reactions are based on chemical differences in the structures of bacterial cell walls. The walls of Gram-negative cells are chemically more complex than those of Grampositive cells. Gram-negative cell wall contains more lipid, polysaccharide, amino acids, and lipoprotein complexes than Gram-positive cell walls. This allows the cell walls of Gramnegative bacteria prone to be decolorized. The chemical components of Gram-positive cell walls however cause them to retain the primary stain and resist decolorization. 2

3 Experiments Precautions: For safety, all biological specimens should be treated as biohazards and Standard Precautions must be STRICTLY applied in handling samples exercised. Latex gloves should be worn at all times to avoid exposure to the infectious agents. All pipette tips and samples should be discarded as bio-hazardous waste. Read the Standard Precautions notice for handling specimens before embarking on the experiment. In this practical, you are provided with two bacteria (X & Y) grown on the same glass slide. You are then asked to prepare and perform the Gram stain, and biochemical tests accordingly. The results are recorded as follows: Tests Bacteria Gram stain Catalase test Staphaurex test Oxidase test X Y I. Preparation of bacterial smear for staining Smear can be prepared from cultures growing on media or directly from swabs collected from sites of infection. In this demonstration, you are asked to help in preparing a smear of bacteria from cultures on solid media, suitable for subsequent staining. 1. Using a glass-writing pen (wax pen) to mark the slide into sections: one section for each smear. 2. With the loop place a small drop of saline on each section of the slide. 3. Flame the loop by holding it vertically in the Bunsen flame until it is heated to redness. Allow the loop to cool momentarily. 4. Holding the loop like a pen, transfer a small amount of the bacterial colony to the saline drop on the slide. 5. Using the loop to mix with saline. 6. Spread the mixture well over the area of the slide. Try to make smear as thin as possible (i.e. almost too thin to be seen when dry). 7. Re-flame the loop to sterilize it. 8. Allow the slide to air-dry completely. 9. "Heat-fix" the slide by passing the slide once through the flame. This ensures the bacteria to firmly adhere to the slide. II. Gram s Staining method Gram stain is the most frequently performed stain in microbiology laboratory. The staining procedure consists of applying a sequence of primary stain (Crystal-violet stain), iodine solution, decolorizer and counterstain (Safranin). The end result is that Gram-positive organisms show a dark purple/blue/black color and the Gram-negative organisms show a pink/red color. 1. Flood the slide with crystal-violet stain, and wait for 15 seconds. 3

4 2. Pour off stain and rinse with iodine solution. 3. Apply iodine solution and wait for 15 seconds. 4. Rinse with running water, and shake off the excess. 5. Decolorize the slide with 95% (v/v) ethanol solution. 6. Rinse with running water. 7. Apply the slide with safranin, and wait for 15 seconds. 8. Rinse with running water, blot dry in air. 9. Examine the slide to distinguish if this bacteria is Gram-positive or -negative. [Hint: oil-immersion objectives may be necessary] III.Laboratory tests for common Gram-positive/negative organisms Many bacteria cannot be identified based on microscopic or cultural characteristics alone, The biochemical properties and reactions of bacteria form the basis for an important series of identification procedures. In each biochemical procedure, the unknown bacterium causes a change of some type in the medium, to which a specific test substance has been added. The change may be indicated by the gas formation, change in color or physical states. For example, the identity of Staphylococci aureus (cause many serious infections) can be determined by performing the following tests: - Macroscopic examination: Chocolate agar - Microscopy examination: Gram stain - Biochemical tests (positive result): catalase test and coagulase test (Staphaurex Test) This can help to differentiate other members of the genus Staphylococci which are normal flora but are coagulase-negative. In this part of practical, you are asked to perform the biochemical tests to confirm the identity of the bacteria X and Y A. Catalase test Catalase test is used to detect enzyme catalase present in bacteria. This enzyme decomposes the H2O2 to release the water and oxygen. This test is often used to differentiate Staphylococci sp. from Streptococci sp.* 2H2O2 (aq) 2H2O (l) + O2 (g) * The genus Streptococci does not produce catalase and consequently is catalasenegative. 1. Using a pipette, deliver a drop of catalase reagent (3% H2O2) on the centre of a 4

5 clean slide. 2. Transfer the bacterial colony from culture using a inoculating needle. 3. Place the colony on the center of a clean cover slip and then over the drop of catalase reagent. 4. Observe any visible bubbling, which if seen indicates the formation of oxygen. B. Staphaurex test The Staphaurex reagent consists of polystyrene latex particles which have been coated with fibrinogen and IgG. When mixed on a slide with a suspension of Staphylococcus aureus, reaction of clumping factor with the fibrinogen, and/or of protein A with the IgG resulted in rapid strong agglutination of the latex particles. 1. Add a drop of Staphaurex reagent on a card provided. 2. Remove a colony with a sterile toothpick and emulsify it in the reagent to make a dense and uniform suspension. 3. Mix in a circular motion. 4. Examine the slide to see if any visible clumping within minutes. C. Oxidase Test Oxidase test is used to determine the enzyme cytochrome oxidase present in bacteria. This enzyme oxidizes an artificial electron acceptor (e.g. tetra-methyl-pphenylenediamine dihydrochloride to form a blue-colored compound (i.e. indophenol blue). Precaution: A false-positive reaction may result if an iron-containing wire is used to transfer the bacteria. It is thus recommended to use platinum wire, disposable loop or wooden sticks to transfer the bacteria. 1. Soak a piece of filter paper in the reagent solution. 2. Use a platinum wire/disposable loop/stick to scrape some fresh growth from the plate, and then rub onto the filter paper. 3. Observe for any blue color formed (within 10 seconds). Result: Positive: blue color indicates oxidase production Negative: no blue color developed Questions: (for discussion) Based on the results of these biochemical tests, what is your conclusion on the types of bacteria X and Y? 5

6 Questions 1. (a). Explain why it is necessary to "heat-fix" the smear before it is stained with Gram stain in the practical. (3 marks) (b). In general practice, the result of Gram stain is important for identification of an unknown bacteria. Explain why. (4 marks) (c). Gram stain can be used to detect fungi. What are the other examination methods commonly used for detection of fungi? Describe ONE of them. (3 marks) 2. (a). List the types of genetic materials identified in the virus? Name ONE example of virus on each type. (4 marks) (b). Describe how the virus infect a cell and cause the cell lysis. (5 marks) (c). Suggest TWO current detection methods of viral infection in a clinical laboratory. (4 marks) 3. (a). What is aspergillosis? Name ONE types of aspergillosis. (3 marks) (b). Illustrated with diagrams, describe how Aspergillus sp. causes the invasive aspergillosis in human. (6 marks) (c). Suggest ONE examination methods commonly used for the diagnosis of an individual infected with malaria. (3 marks) 6