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  • 1. Introduction to Basic Bacteriology TRANSFERRING OF BACTERIAL COLONIES USING ASEPTIC TECHNIQUE. INTRODUCTION TO BACTERIAL STAININGIntroductionAseptic TechniqueA strictly sterile (aseptic) procedure is essential when collecting the specimen from the sitesthat are normally sterile such as collection of blood, cerebrospinal fluid (CSF), or effusions.An aseptic technique is used not only to prevent contamination of the specimen but also toprotect the patient and worker.Microbiologists and students of microbiology may use an aseptic technique to attempt tokeep specimens of microorganisms free of microbial contamination. People may commonlyuse the aseptic technique when transferring bacteria from one test tube or flask to another.This method of preventing unwanted microorganisms from gaining access is termed asASEPTIC TECHNIQUE.Inoculating Loops (Bacteriological Loops) and NeedlesThe inoculating loop is sterilized by passing it through the flame of a gas burner at an angleuntil the entire length of the wire becomes orange due to the heat. In this way allcontaminants on the wire are incinerated. It is imperative that you can incinerate the entirewire to ensure absolute sterilization. The shaft should also be briefly passed through theflame to remove any dust or possible contaminants. To avoid killing the cells and splatteringthe culture, cool the inoculating wire by tapping the inner surface of the culture tube or theedge of uninoculated agar or medium prior to obtaining the inoculum.Preparation of SmearAvoidance of thick, dense smears is absolutely essential. A good smear is one that, whendried, appears as a thin translucent whitish layer or film. Those made from broth cultures orcolonies from a solid medium require a variation in technique.Broth Culture:One or two loopfuls of suspended cells should be applied directly to the centre of a cleanglass slide with a sterile inoculating loop and spread evenly over an area about the size of a10 cent coin.Allow this thin smear to completely air dry.Cultures from a solid medium:Aseptically remove a small amount of the culture from the agar surface with a sterile loopand just touch it several times to a drop of sterile water on a slide until it just turns cloudy. 1|Page
  • 2. Burn the remaining bacteria off of the loop. (If too much growth is emulsified the smear willbe so dense that you won’t be able to see stained individual bacterial cells).Using the loop, spread the suspension to form a thin smear about a 10 cent coin in size.Allow this thin smear to completely air dry.Heat FixationBefore staining bacteria, you must first understand how to "fix" the organisms to the glassslide. If the preparation is not fixed, the organisms will be washed off the slide duringstaining. A simple method is that of air drying and heat fixing. The organisms are heat fixedby rapid passage of the air-dried smear two to three times over the flame of a Bunsen burner.The heat coagulates the organisms proteins causing the bacteria to stick to the slide.Caution: Too much heat might distort the organism and, in the case of the Gram stain, maycause gram-positive organisms to stain gram-negatively. The slide should feel very warm butnot too hot to hold.StainingSince bacteria are almost colorless and therefore show little contrast with the broth in whichthey are suspended, they are difficult to observe when unstained. Staining microorganismsenables one to: see greater contrast between the organism and the background, differentiate various morphological types (by shape, arrangement, gram reaction, etc.), observe certain structures (flagella, capsules, endospores, etc.).In order to understand how staining works, it will be helpful to know a little about thephysical and chemical nature of stains. Stains are generally salts in which one of the ions iscolored. (A salt is a compound composed of a positively charged ion and a negativelycharged ion.) For example, the dye methylene blue is actually the salt methylene bluechloride which will dissociate in water into a positively charged methylene blue ion which isblue in color and a negatively charged chloride ion which is colorless.Dyes or stains may be divided into two groups: basic and acidic. If the color portion of thedye resides in the positive ion, as in the above case, it is called a basic dye (examples:methylene blue, crystal violet, safranin). If the color portion is in the negatively charged ion,it is called an acidic dye (examples: nigrosin, congo red).Because of their chemical nature, the cytoplasm of all bacterial cells have a slight negativecharge when growing in a medium of near neutral pH. Therefore, when using a basic dye,the positively charged color portion of the stain combines with the negatively chargedbacterial cytoplasm (opposite charges attract) and the organism becomes directly stained. Anacidic dye, due to its chemical nature, reacts differently. Since the color portion of the dye ison the negative ion, it will not readily combine with the negatively charged bacterialcytoplasm (like charges repel). Instead, it forms a deposit around the organism, leaving theorganism itself colorless. Since the organism is seen indirectly, this type of staining is calledindirect or negative, and is used to get a more accurate view of bacterial size, shapes, andarrangements. 2|Page
  • 3. StreakingStreak the loop across the surface of the agar medium to obtain single isolated bacterialcolonies originating from a single bacterium or arrangement of bacteria. In order to avoiddigging into the agar as you streak the loop over the top of the agar you must keep the loopparallel to the agar surface. Always start streaking at the "12:00 clock position" of the plateand streak side-to-side as you pull the loop toward you. As you follow, each time you flameand cool the loop between sectors, rotate the plate counterclockwise so you are alwaysworking in the "12:00 clock position" of the plate. This keeps the inoculating loop parallelwith the agar surface and helps prevent the loop from digging into the agar.EXPERIMENT 1: Transferring Of Bacterial Colonies Using Aseptic TechniqueIn the future, every procedure in the lab will be done using these similar aseptic techniques.This procedure for aseptically transferring microorganisms is as follows:1.1 Sterilize the inoculating loop. The inoculating loop is sterilized by passing it at an angle through the flame of a gas burner until the entire length of the wire becomes orange from the heat. In this way all contaminants on the wire are incinerated. Never lay the loop down once it is sterilized or it may again become contaminated. Allow the loop to cool a few seconds to avoid killing the inoculums.1.2 Remove the inoculums. (to be done in pair) You are provided with organisms Staphylococcus epidermidis (labeled A) and Klebsiella pneumoniae (labeled B) growth on a nutrient agar (NA) plate. A. Transferring the inoculum from a plate dish (organisms growing on an agar surface in a Petri plate) into a broth tube Sterilize the inoculating loop in the flame of a gas burner until it is orange. Allow the loop to cool a few seconds Lift the lid of the culture plate slightly. Scrape off a small amount of the organisms labeled A. Pick up a sterile nutrient broth tube and remove the cap with the little finger of your loop hand. Do not set the cap down. Briefly flame the lip of the broth tube Place the loopful of inoculum into the broth, and withdraw the loop. Do not lay the loop down on the bench! Again flame the lip of the tube. Replace the cap Repeat the above procedure for the organism labeled B. Resterilize the loop by placing it in the flame until it is orange. Now you may lay the loop down until it is needed again. Incubate the broth in 37°C incubator for 1 hour. After 1 hour incubation, use this broth for the Experiment 1.2B 3|Page
  • 4. B. Transferring inoculum from a broth culture (organisms growing in a liquid medium) to the solid medium surface (Nutrient Agar) and Streaking Labeled your broth tube as A or B. Use this broth later for the experiment 2.2 Hold the culture tube in one hand and in your other hand, hold the sterilized inoculating loop Remove the cap of the pure culture tube with the little finger of your loop hand. Never lay the cap down or it may become contaminated. Very briefly flame the lip of the culture tube. This creates a convection current which forces air out of the tube and preventing airborne contaminants from entering the tube. Keeping the culture tube at an angle, insert the inoculating loop and remove a loopful of inoculum. Again flame the lip of the culture tube. Replace the cap Transfer the inoculum to the agar surface. Proceed with streaking as the instruction given below Lift the edge of the lid just enough to insert the loop. Streak the loop across the surface of the agar medium. The streaking allows you to obtain single isolated bacterial colonies originating from a single bacterium or arrangement of bacteria Resterilize the inoculating loop* In the future, every procedure in the lab will be done using similar aseptictechniqueEXPERIMENT 2: STAINING (to be done individually)Prepare two smears on two different slides of the normal flora and cells of your mouth. Useone for simple staining and another one for the differential staining.Procedure Using a sterile cotton swab, vigorously scrape the inside of your mouth and gums. Rub the swab over the slide ( do not use water)2.1 Simple Staining a. Heat-fix the smear b. Stain with crystal violet for one minute. c. Wash off the excess crystal violet with water. d. Blot dry and observe using oil immersion microscopy. 4|Page
  • 5. 2.2 Differential Staining Do smears on different slides and proceed with Gram staining Student 1 – From plate labeled A Student 2 – From plate labeled B Per Bench Student 3 – From incubated nutrient broth labeled A Student 4 – From incubated nutrient broth labeled B Gram Stain: The most important stain used in bacteriology, Procedure Air dry and heat fix. Flood the fixed smear with crystal violet for 1 min. Wash off the stain with tap water. Flood the smear with Lugol’s iodine for 1 min. Then wash off with tap water. Decolourize rapidly with iodine-acetone and immediately wash with tap water. Lastly flood the smear with dilute carbol fuchsin for 1 min. Then wash with water. Wipe the back of the slide, and place the smear on the bench to air dry. Examine the smear microscopically, first with the 10X objective to see the distribution of smear and then examine the smear with oil immersion by using the 100X objective. Result: the stain divides bacteria into 2 groups Gram positive bacteria stain a purple colour Gram negative bacteria are stained redCAVEATS Remove any immersion oil from the oil immersion lens (x100 Objective) before putting the microscope away using lens paper. (Paper towel or kim-wipes may scratch the lens.) 5|Page
  • 6. DEMO SLIDE2.3 Negative StainingIn negative staining, the negatively charged color portion of the acidic dye is repelled by thenegatively charged bacterial cell. Therefore the background will be stained and the cell willremain colorless. Procedure a) Place a drop of Indian Ink toward one end of the slide. b) Aseptically place a loopful of the inoculum of Klebsiella pneumoniae into the drop of stain and mix with the loop c) Place a slide against the drop of suspended organisms and allow the drop to spread along the edge of the applied slide d) Push the slide away from the previously spread drop of suspended organisms, forming a thin layer. e) Air dry the smear and then observe using oil immersion microscopy. 6|Page