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TEB Microscopy of bacteria Related Topics Bacteria, sterility, microscope, and staining. Principle The microscopic examination of microorganisms can be performed based on live specimens or in fixed and stained preparations. Live specimens of microorganisms are prepared in a drop of water (or culture liquid). The aim of staining bacteria preparations is to increase the contrast between the bacteria and their environment from which they hardly stand out when unstained. Equipment 11Culture vessel 64834-00 1 Scissors, straight, blunt, l = 140 mm 64625-00 1 Glass beaker, DURAN®, tall, 100 ml 36002-00 1 Microscope slides, 50 pieces Cover glasses, 18 mm x 18 mm, 50 1 pcs. Inoculation loop with holder, l = 230 1 mm 1 Water, distilled, 5 l 64691-00 1 Immersion oil, 50 ml Glass rod, boro 3.3, l = 300 mm, d = 10 7 mm Safety gas tubing, DVGW, sold by 1 metre Bunsen burner with stop cock, for natural gas SWIFT student microscope 1 M3602C-3 1 Staining tweezers, after Kühne 1 32167-05 63021-99 64726-00 64796-00 31381-05 1 Petri dish, d = 200 mm, glass Graduated cylinder, 100 ml, BORO 1 3.3 Filter paper, 580 mm x 580 mm, 10 1 sheets 1 Carbol-fuchsin solution, 100 ml 40485-05 1 Entellan for microscopy, 100 ml 31294-10 1 Ethyl alcohol, absolute, 500 ml Methylene blue B, for microscopy 25 1 g 30008-50 39281-10 64685-00 64936-00 31246-81 36629-00 32976-03 31463-10 31567-04 Fig. 1: Staining of bacteria preparations. www.phywe.com P4140300 PHYWE Systeme GmbH & Co. KG © All rights reserved 1 TEB Microscopy of bacteria Tasks Examine a bacteria preparation under a microscope and apply the standard methods that are described herein. Procedure Procurement of microorganisms for microscopic examinations A quick way to obtain microorganisms for microscopic examinations, e.g. in order to identify and observe different forms of bacteria, the existence or non-existence of proper motion, the formation of spores, etc. in living or fixed and stained preparations, is an infusion of plant parts. Infuse a hand full of grass, hay, or lettuce leaves in tap water in a culture vessel and let this infusion stand uncovered at room temperature. The water level should be several centimetres above the plant parts and approximately three quarters of the culture vessel should be filled Fig. 2: Hay infusion in a glass. (Fig. 2). Large, cumbersome plant parts can be broken up beforehand. Within three to five days, a biofilm forms on the liquid surface. At first, it consists solely of bacteria. Later, after one to two weeks, it includes also various types of microscopic fungi next to protozoa (mainly ciliates). The reason for the formation of the biofilm is that the plant parts that are filled into the culture vessel are infected with microorganisms and their spores. The addition of water leads to excellent culture conditions, which is why they multiply rapidly. They grow in the form of a biofilm since they are mainly aerobic microorganisms, which means that they can develop only under sufficient oxygen partial pressure. At the surface of the liquid, the oxygen level is the highest. Observation of microorganisms with the aid of a live specimen Transfer a drop of water onto a microscope slide. Flame an inoculation loop in the flame of a gas burner and let it cool. Then, use it to withdraw some of the microorganisms to be studied, e.g. from the biofilm on an infusion of plant parts or from a colony that has developed in a Petri dish, and stir them carefully into the drop of water (Fig. 3). Do not use an excessive amount of material, since otherwise a compact mass of close-packed microorganisms would result. In such a mass, details are difficult to Fig. 3: Transfer of bacteria onto a microscope slide. discern. Only a very slight, milky turbidity should form in the drop of water. This can be checked rather easily by placing the microscope slide on a dark surface. Flame the inoculation loop again in the flame of a gas burner until it is red hot in order to clean and sterilise it. Cover the preparation with a cover glass and place it under the microscope with a 50 times microscopic magnification (objective 10x, eyepiece 5x). Depending on the size of the objects, examine it with a 400 and 1000 times magnification (objective 40x or 100x, eyepiece 10x). In many cases, however, the bacterial cells are so large that it is not necessary to use an immersion objective. Microorganisms that are already distributed in a nutrient solution are usually transferred to a microscope 2 PHYWE Systeme GmbH & Co. KG © All rights reserved P4140300 Microscopy of bacteria TEB slide, which is then covered with a cover glass, without using a drop of water. Unstained living preparations of microorganisms have a relatively low contrast. The cells or union of cells hardly set themselves apart from their environment. The contrast of the preparation can be increased by closing the iris diaphragm of the illuminator of the microscope by half or two thirds. Fixing of bacteria preparations The internal structures of most types of bacteria are so small that they cannot be resolved by a light microscope, which means that they are not visible. This is why the conservation of the internal structures of the cells is usually refrained from and the bacteria preparations are fixed by heat. Flame an inoculation loop in the flame of a gas burner and let it cool. Then, use it to withdraw some of the bacteria mass, e.g. from the biofilm on an infusion of plant parts and stir it as evenly as possible into a drop of water on a microscope slide. The quantity of the bacteria mass is correct if the Fig. 4: Fixing of bacteria preparations. drop of water shows a slight milky turbidity when viewed on a dark surface. Flame the inoculation loop again in the flame of a gas burner until it is red hot in order to clean and sterilise it. After the preparation has dried completely in the air, pass the microscope slide, with the coated side up, three times for approximately one second for each pass through the non-bright and non-crackling flame of a gas burner (Fig. 4). During this process, the microscope slide reaches a temperature of approximately 120°C. As a result, the bacterial cells are killed while preserving their outer shape and they are fixed on the microscope slide. Staining of bacteria preparations Nearly all stains are based on the electro-adsorption of dye ions to free valences of the molecules that are involved in the formation of the bacterial cell. To a lower degree, stains are also based on the affinity of a dye for certain parts of the bacterial cell or on chemical reactions inside the cell if the reaction product is a dye. Prior to staining, however, the bacteria must be fixed. For staining, the fixed preparation is placed on a staining bench above a staining tray that can be assembled rather easily. Use half a Petri dish with a diameter of 200 mm. Attach four medium-sized paper clips to the rim of the dish approximately in the middle so that two pairs of paper clips face each other from opposite ends of the dish. Then, push two glass rods (300 mm long, diameter 5 mm) through the eyelets that are formed by the paper clips and push the paper clips down in order to fix the glass rods in place. Drop some staining solution onto the microscope slide so that the fixed bacteria mass is completely covered. After the staining time, let the staining solution flow into the staining tray by tilting the microscope slide. Then, rinse the preparation under tap water until no more dye flows off. Dry it in the air by placing it on some filter paper and by tilting it against the staining tray. The stained preparations can be viewed with 400x and 1000x magnification (objective 40x or 100x, www.phywe.com P4140300 PHYWE Systeme GmbH & Co. KG © All rights reserved 3 TEB Microscopy of bacteria eyepiece 10x). If an immersion objective is used, they can also be examined without a cover glass. If, however, they are to be stored as permanent preparations, add a drop of embedding agent (Entellan) to the microscope slide and cover it with a cover glass. For bacterial preparations, the following standard dyes can be used: Diluted carbol-fuchsin solution Prepare a 1:5 dilution of a carbol-fuchsin solution according to ZIEHL-NEELSEN in distilled water. This means that four parts of distilled water are added to one part of staining solution. The staining time is 5 minutes. The bacteria are stained bright red. Diluted methylene-blue solution Prepare a 1:5 dilution of a saturated, alcoholic methylene-blue solution in distilled water. This means that four parts of distilled water are added to one part of staining solution. The staining time is 5 minutes. The bacteria are stained in a deep blue. 4 PHYWE Systeme GmbH & Co. KG © All rights reserved P4140300