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VCLab 5 Spore stain and acid fast stain SPORE OR ENDOSPORE STAIN: When the environment becomes too harsh to survive, some bacteria have the ability to eliminate all their cytoplasm and condense all their essential DNA and organelles into a highly resistant structure called a spore, which is metabolically inactive. When the environment improves, they can re-establish themselves. Only sterilization can kill a spore. Spores are usually only produced by bacillus bacteria that are found in the soil, such as Bacillus (non-pathogenic) and Clostridium (tetanus and botulism) a. b. c. d. PRIMARY STAIN: Malachite green MORDANT: Heat (allows dye to penetrate the spore) DECOLORIZER: Water COUNTERSTAIN: Safranin ENDOSPORE STAINS Endospores are the most resistant cells on the planet. Whatever the conditions are for killing spores are the conditions necessary for sterilization. The reason we use an autoclave at 121°C for at least 15 minutes with steam under pressure is because that is what it takes to kill endospores. If there was no such thing as an endospore, we could just boil everything for ten minutes to get sterilization. Don’t confuse endospores with other spores like reproductive spores of fungi. Reproductive spores are not resistant. Bacterial endospores are not reproductive. Terminology Vegetative cell: This is a cell that can make the endospores, but they are not present yet. Endospores can be inside the cell or free. Free endospores do not have any cytoplasm left. Two major genera that produce endospores 1. Bacillus (an obligate aerobe; must have O2 2. Clostridium (obligate anaerobe; must not have any O2) (There are other bacteria that are aerotolerant aerobes; if they are exposed to air, they won’t die, but they won’t grow, either.) Why make endospores? To resist adverse environmental conditions: changes in temperature, pressure, pH, oxygen, and moisture. When we inoculate media and place it in the incubator, that’s all the nutrients the bacteria get. No one comes in and adds more vitamins. When the nutrients are all used up, the bacteria die unless they can make endospores. Characteristics of Endospores 1. Highly resistant to adverse environmental conditions 2. No metabolism 3. No water left in the cell 4. They retain their DNA 5. They have a vary thick spore coat for protection 6. Can remain viable (able to return to the vegetative state and reproduce again) for millions of years Two Cycles of Endospores 1. Vegetative Cycle 2. Sporulation Cycle VEGETATIVE CYCLE Endospore-forming bacteria reproduce by binary fission, just like all other bacteria. Every 15-20 minutes, they split into 2, then 4, then 8, then 16, etc. Vegetative cells are happy, they have nutrients, and environmental conditions are good. Therefore, they have no endospores. In a vegetative state, these cells are easily killed by heat or chemicals. When the environmental conditions become adverse, the nutrients deplete, the O2 levels either go up or down (whichever is unsatisfactory for that organism), and the water availability goes down and becomes dry (dessication). Under such conditions, the vegetative cell will then enter into the sporulation cycle. We can induce this cycle by taking bacillus and incubating them for 48 hours, because the nutrients will deplete by then. After 72 hours, there will only be free endospores left. In 24 hours, we can see vegetative cells with and without endospores, as well as free endospores. SPORULATION CYCLE We can see endospores even without stain because they are highly refractive; light from the microscope bounces off of it. Spore Locations Within Cell 1. Central 2. Terminal 3. Subterminal Today, we will use Bacillus spp, which means that we don’t know what species are in there. These were obtained from your air exposure plates. We know they are not clostridium, because the incubator allows air to get in. There are two endospore stain techniques we will use today. 1. SHAEFFER-FULTON SPORE STAIN Clean two or three slides with Bon ami, rinse, and clean with alcohol. Add one loop of water to the slide and add a needle sample of Bacillus from the culture tube. Air dry, heat fix, and prepare the steam heat like we did last lab period. Place a small square of bibulous paper over the smear, and this time add one drop of malachite green every 30 seconds for 5 minutes. Endospores and cytoplasm will now be green. You need a spore stain to see free endospores. Rinse off the stain, and the cytoplasm will be clear. Counterstain with safranin to stain the cytoplasm. a. Primary Stain: Malachite green b. Mordant: Steam heat c. Decolorizer: distilled water d. Counterstain: Saffranin 2. DORNER METHOD For this method, we will have to make a spore suspension: a. Place 5 drops of sterile water in a test tube (one tube per table). b. Add several loopfuls of bacillus to the tube of water. c. Add 5 drops of carbolfuscian. d. Heat in a beaker of boiling water for 10 minutes. e. Cool. f. Mix several loopfuls of bacteria from the suspension in one drop of nigrosine on the slide. g. Either spread the nigrosine with another slide or smear the drop into a large circle with your loop. h. After you sterilize your loop for the last time, wipe it off with a paper towel to remove the nigrosine. i. Air dry, do not heat fix. j. Examine under oil The cells will look clear with a red endospore inside. You may also see free endospores stained red. ACID-FAST STAIN The results of this stain are recorded as acid-fast or non acid-fast. An example is the ZiehlNeelsen stain. Acid-fast bacteria look pink and non acid-fast look blue. 1. 2. 3. 4. PRIMARY STAIN: Carbol fuchsin (purplish-pink color) MORDANT: heat DECOLORIZER: acid alcohol COUNTERSTAIN: Methylene blue This is the stain of choice if one suspects an organism with a cell wall made of mycolic acid, which is a waxy substance that resists Gram stains. The heat in this procedure will melt down the wax in the cell wall to allow the stain to get in. Two organisms that are acid-fast that are pathogens (cause disease) are Mycobacterium and Nocardia. MYCOBACTERIUM 1. Mycobacterium tuberculosis: an air-borne pathogen that causes tuberculosis. 2. Mycobacterium leprae: Causes Hansen’s disease (formerly known as leprosy). NOCARDIA 1. Nocardia asteroides: lives in the soil. When inhaled, it can cause pneumonia, but usually only an opportunistic infection in immunocompromised patents. Opportunistic infections are infections caused by organisms that usually do not cause disease in a person with a healthy immune system, but can affect people with a poorly functioning or suppressed immune system. They need an "opportunity" to infect a person. Immunocompromised patients include elderly people or infants, AIDS or HIV-infection, Immunosuppressing agents for organ transplant recipients, chemotherapy for cancer patients, malnutrition, medicines (some antibiotics), medical procedures (surgeries, especially implanted joint replacements or internal fixation hardware such as screws and plates for broken bones). ZIEL-NEELSEN ACID-FAST STAIN The acid fast stain is a differential stain used to identify only two types of bacteria. The only organisms that are acid fast (the acid-fast genera) are: 1. Mycobacterium a. Causes Hansen’s Disease (leprosy) b. Causes Tuberculosis (TB) c. There are also non-pathogenic species 2. Norcardia (opportunistic pathogens; only cause disease in those with poor immune systems, etc). The thing that makes organisms acid fast is the wax (mycolic acid) in their cell walls. Their endospores resist stain because of mycolic acid. Clinically, it is important to be able to identify these organisms quickly. This test is only used when a patient is suspected of having TB or leprosy. It is especially useful when someone is suspected of having TB; the sample is obtained from the sputum, and an acid-fast stain is performed to give a preliminary diagnosis right away. It can also be performed on patient samples to track the progress of antibiotic therapy and determine the degree of contagiousness. There are 10 million new cases of TB per year, 3 million deaths, and it affects 1/3 of the world’s population. The results are recorded as AF (acid fast) or NAF (non-acid fast). Don’t record them as positive or negative (like the lab manual says) or you may get them confused with Gram stain results. In the SF stain technique, the primary stain is applied with heat (steam). The reason for this is that the heat increases solubility of the mycolic acid so it can react with the primary stain. Therefore, heat allows stain to penetrate resistant cells because mycolic acid is waxy. Blotting paper must be used on top of the stain when heat steam is used; it keeps the stain from drying out. We will be preparing a slide that is a mixture (emulsion) of two organisms: 1. Mycobacterium smegmatis (AF) 2. Staphylococcus aureus (NAF) Mycobacterium smegmatis is not pathogenic (we are only SL-2 here). It is saprobic. That means it lives off dead organic matter. It is part of the normal microbiota of our skin and the oils in our skin. It also likes dirt. So if you don’t wash regularly, this organism will thrive. It especially lives on the external genitalia: under the foreskin of uncircumcised males and the labia majora of females. It produces a cheesy substance called smegma, which has a foul odor. It only takes one day without washing for it to grow. Hospitalized patients nowadays are in such bad shape they can’t take care of themselves very well, so they frequently get smegma. You’ll learn to recognize the smell! With the ZN technique: 1. Primary stain is carbolfuscian (lipid soluable; can penetrate waxy cell wall). It will stain both AF and NAF. 2. Mordant is the steam heat 3. Decolorizer is ACID alcohol. This will rinse the color out of the NAF only. 4. Counterstain is methylene blue. This will be taken up by the NAF cells. Ziel-Neelsen Acid Fast Stain Technique 1. Get out a hot plate (the kind that has a coil) and set it on high. 2. Get a metal beaker from your tote box and fill it half way with tap water; place on the hot plate. 3. Place a wire stain rack on top of the beaker. 4. Place a wire mesh square on top of the stain rack. 5. Clean one slide. 6. Prepare an emulsion: a. Place one loopfull of water on the slide. b. Remember to grasp the culture tubes by the glass, not the cap! c. Add one needle sample of Mycobacterium smegmatus. This organism is very waxy, so you have to tap and mix VERY WELL to break it up completely. Otherwise, it will clump on your slide. Page 92 in your manual shows a slide that is clumpy. d. Add one needle sample of Staphylococcus aureus and tap and mix. 7. Air dry completely to avoid aerosols. 8. Heat fix. 9. Cut a SMALL square of bibulous paper that is the size of your smear. Place this square directly on your slide. This keeps the smear from drying out. 10. When the steam starts showing from the beaker, place the slide on the wire mesh and add ONE DROP of carbolfuscian (the primary stain) to the paper every 30 seconds for five minutes. Too many drops will cause the carbolfuscian to drip into the water beaker and boil. This will release phenol from the stain, which is a dangerous aerosol. After five minutes, turn off hot plate. 11. When the slide is cool, pick up slide with a clothespin. 12. Throw out bibulous paper into the regular trash container. 13. Rinse gently with distilled water. 14. Decolorize with acid alcohol (in the stain kit). Do NOT use regular alcohol! Apply the acid alcohol with a rock-and-roll agitation for a few seconds until the color rinses clear. 15. Rinse gently with distilled water to stop the decolorization process. 16. Take the slide to the sink and counterstain with methylene blue for one minute. 17. Rinse gently with distilled water. 18. Blot dry. 19. Observe under 1000x and oil. 20. Look for individual cells. You will see AF rods that are purple-pink, and little blue cocci. This mixture is what you would actually see clinically, because a patient will not have a pure culture. What does a slide of ZN look like? What does a slide of SF look like?