Lab 4&5 Isolation of Pure Culture & Staining G+ve and G-ve PDF

Basic Microbiology Practical No.: 4 Isolation of pure culture and Gram staining. Intended Learning Objectives (ILOs): By the end of this lab the student shall be able to: 1. Able to define the pure culture and how to obtain it. 2. Able to explain the purpo...

Basic Microbiology Practical No.: 4 Isolation of pure culture and Gram staining. Intended Learning Objectives (ILOs): By the end of this lab the student shall be able to: 1. Able to define the pure culture and how to obtain it. 2. Able to explain the purpose of different isolation techniques. 3. Able to demonstrate how to perform an isolation streak plate procedure. 4. Able to prepare bacterial smears and perform Gram stain 5. Able to explain the chemical basis and principle of the staining technique. 6. Distinguish between the Gram positive and negative bacteria under the light microscope. 7. Describe the differences between the cell wall structures of gram negative and gram-positive cells. 8. Understand clearly the use of different media for isolation of different bacterial organisms. Precaution: 1. Wear appropriate personal protective equipment for the laboratory procedure. 2. All samples must be processed in line with SOP designed for the test. 3. Result must be recorded immediately. Introduction/ Background: Culture: Microorganisms that is cultivated in the lab for the purpose of studying them. It is important to keep in mind that most of the microbiological samples comes from non-sterile sites, i.e. mixed culture. Therefore, it is very crucial in Page 1 microbiology lab to obtain pure culture, in order to be able to identify or diagnosis the cause of the infection. Pure Culture: A pure culture contains a single or one type of bacterial species. Methods of isolation of pure culture: 1. Streak plating on solid agar medium using a sterile inoculation loop. 2. Spread plating on solid agar medium with a glass spreader 3. Pour plating of diluted samples mixed with melted agar medium q The main purpose of streak, spread and pour plating is to isolate individual bacterial colony on an agar medium. q Spread and Pour plate methods are used also to enumerate colonies, which represented as Colony Forming Units (CFU). 1. STREAK PLATE TECHNIQUE STREAKING: q It is a technique that is used to spread a clinical sample or bacteria out on the surface of a growing medium (agar plate) so that individual bacterial colonies can be isolated and examined further. q The streak plating technique isolates individual bacterial colony (CFU) on the surface of an agar plate using a wire loop. Purpose: Streaking method is used in order to check - 1. whether a culture consists of only one type of organism or species (pure culture) or if there are more than one species in it (contaminated or mixed) 2. Whether a culture is viable, i.e. able to grow. Page 2 Principle: q The principle is that individual microbial cells can be separated by dragging them over the surface of the agar, and then given a chance to grow into individual colonies. q Streak plate is widely used method to separate individual bacteria from mixtures. Precautionary note: Ø Try to follow the pattern as closely as possible. Creativity is a good thing in some situations, but not when doing an isolation streak plate. Ø Touch your loop gently to the agar. Try not to cut the surface of the plate. Ø Keep your plate in the incubator covered with the lid in an upside down position. 2. SPREAD PLATE TECHNIQUE Principle: A specific amount of a liquid sample or culture placed on an appropriate agar and spread using glass spreader. A perfect spread plate technique will result in evenly distributed number of colonies that is visible and countable. Microbiological Staining Microbial cells are very small in size and cannot be seen with unaided eyes. Therefore, it is essential in microbiology to use microscopes in order to help us in the identification of the bacterial cellular morphology and behaviour. However, microbial cytoplasm is usually transparent, thus, it is important to apply some stains on the microorganisms before they can be viewed with the light microscope. Types of Staining techniques: 1. DIRECT STAINING: Aniline dyes are able to stain bacterial cell directly. Exceptions are bacterial spores, e.g., of Bacillus spp. and the bacteria that have waxy coating on their cell wall, e.g., Mycobacterium spp. Page 3 2. INDIRECT OR NEGATIVE STAINING: These are the dyes which stain only the background, e.g., Nigrosin or India ink used either for observing mucilaginous covering enveloping bacteria (capsules) or certain spores of fungi or cells of unicellular animals. 3. SELECTIVE STAINS: These stains are used for staining particular parts of the organism such as spores, metachromatin granules, flagella, nuclei, etc. 4. DIFFERENTIAL STAINS: These stains are those which enable one to differentiate two different groups of bacteria in a mixture, for instance, gram-positive and gram- negative. (e.g. Gram staining & ZN staining) GRAM STAINING Hans Christian Gram (1853–1938), developed the Gram Staining technique in 1882. 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 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. Page 4  Gram-positive cells will appear blue to purple, while Gram-negative cells will appear pink to red. Page 5 PRACTICAL CASE #1 A 32 years old female patient was admitted to Prince Mohammed Bin Nasser hospital for surgery. A few days later after the surgery, she noticed an area of inflammation around the site of the wound which began to ooze pus. The doctors send a swab of the pus to the lab. DAY 1: Isolation of pure culture and preliminary identification of the organism PART-A: Isolation of pure culture Materials: Equipment: Safety cabinet, Bunsen burner or incinerator, inoculation loop, incubator Materials: 1x Blood agar plates, 1x MacConkey agar plates (one of each agar per student) Specimens: Swab sample METHOD: 1. Using the loop, take a drop of the clinical sample (Culture) provided and streak it carefully in a line across the surface of the agar (First streaking area Q1). With the same loop, a 2nd, 3rd and 4th line drawn parallel to the first. Close the lid of the Petri dish immediately. (see the figure below) 2. Sterilize the loop in the flame once again, and allow it to cool. 3. Turn the Petri dish so that the end of the previous lines can be the start of the next ones. 4. Take a cooled loop and make 2 or 3 streaks as before (on the following streaking area Q2). Close the lid of the Petri dish immediately. Repeat step 2, 3, and 4 two more times for the Q3 and Q4 area, then finish off with a single zigzag streak across the middle. Page 6  q Seal and Label the Petri dish with the culture reference, name, date, sample, and condition of incubation. Place it in an inverted position in the incubator at 37 0C for 24-48 hours. PART-B: Gram stain *ONE SLIDE PER STUDENT Materials: Equipment: Bunsen burner, cleaned microscope slide, Water, Slide dryer, loop Control: Agar plate cultures of P. aeruginosa and Staphylococcus aureus. Specimens: Sample A and B (one sample per student) Reagents: Gram staining consists of four components: o Primary stain (Crystal violet or Gentian violet) o Mordant (Gram's Iodine) o Decolourizer (acetone or Ethanol) o Counter stain (safranin or neutral red) METHODS: Slide preparation: 1. Divide the slide into three section as the diagram below 2. Prepare the smear by placing a small drop of water on the slide. 3. With an inoculating loop, pick one colony (Direct specimen or culture to be taken); place colony in the drop of water and make a milky suspension. Page 7  Note: if the sample is from liquid culture, it does not require a drop of water on the slide 4. Leave for air dry 5. Heat-fix the smear by passing the slide over the burner flame several times (2-3 times). Take care not to overheat (overheating will lyse the cells). Test the temperature by placing the slide on the back of your hand. If it’s too hot for your hand, it’s too hot for the bacteria. P. aeruginosa S. aureus Sample A or B Steps of Gram Staining: (See figure below) 1. Flood the slide with crystal violet stain and leave for 45-60 seconds 2. Pour off the crystal violet stain, Wash with running tap water 3. Flood the slide with Gram’s iodine for 45-60 seconds 4. Flood the slide with decolorizer (95% acetone/alcohol) watching for the dye to continue washing from the cells (usually for 4-6 seconds. Be careful not to overdo this step. 5. Wash under gentle running tap water. 6. Flood the slide with counter stain Safranin for 45-60 seconds 7. Wash with running tap water and allow the stained smear to dry using filter paper. 8. Observe under the microscope Note: Start gradually from the lowest magnification power without oil immersion. Add oil ONLY with x100 lens. Page 8 Observation Gram reaction Shape Arrangement Sample A Sample B References: 1. Bharti Arora, D. R. Arora (2015): Practical Microbiology. ISBN: 81-239-14095-9. Page 1 – 218. 2. Steven Obenauf, Susan Finazzo (2016): Microbiology Fundamentals. A Clinical Approach. Page 1 – 318. Page 9