Explore 6. STAIN procedure o Place slide into container of crystal violet, leaving in for 1 minute.

Wash WELL with water. o Place slide into container of Gram's iodine, leaving in for 1 minute. Wash WELLw ith water. o Flood acetone-alcohol quickly on the slide, and wash off within 5-10 seconds(fro m beginning of decolorizer added). Wash WELL with water. o Place slide into container of safrinin, leaving in for 1 minute. Wash WELL withw ater. o Blot dry with bibulous paper.7. Focus on smear using low power lens, ending up o n 100X oil immersion. Be sure thatyou have a drop of oil on the slide before rot ating your 100X objective lens into place. Page 39 LABORATORY REPORT SHEET QUESTIONS:1. Critiquing your gram stain technique: o Are the cells well distributed on the slide? o Are the cells stained uniformly and is the gram reaction correct? o Is the arrangement of the bacterium consistent across the various fields of visi on?2. What are the gram reaction, shape, and arrangement of Bacillus ?What are the gram reaction, shape, and arrangement of E. coli ?3. What color is E. coli when gram stained? Name the dye that gives it this color.4. To what cell structu re do the 2 dyes bind?5. 2. List at least 3 differences between gram positive an d gram negative bacteria.6. Would it be useful to perform a gram stain on a mixe d culture? Why?7. If a gram stain gives you some valuable information on feature s of the bacterium,what would be the benefit of ever performing a simple stain? Page 40 THE ENDOSPORE STAIN Endospore production is a very important characteristic of some bacteria, allowi ng them to resistadverse environmental conditions such as desiccation, chemical exposure, extreme heat, etc.Bacterial endospores are the most resistant structur es of all living organisms, and they can livein this dormant dehydrated state fo r hundreds and hundreds of years (even some documented atthousands of years). En dospores are not for reproduction: One spore forms inside of thevegetative cell. When the spore germinates, one vegetative cell will be produced. The stimulusfo r sporulation can vary nutrient depletion, desiccation, chemicals, heat, etc.As a sp ore forms inside of the vegetative cell, the spore wall chemically changes and t hicken.This sporulation process changes the spore s stainability, making it increasi ngly resistant to thestaining dyes, and so a gimmick steaming---enhances the primary dye s penetration. The primary dye malachite green is a relatively weakly binding dye to the cell wall and spore wa ll. Infact, if washed well with water, the dye comes right out of the cell wall, however not from thespore wall once the dye is locked in. That is why there doe s not need to be a

decolorizer in thisstain: it is based on the binding of the malachite green and the permeabi lity of the spore vs. cellwall. Spores will be a light green: Vegetative cell wa lls will pick up the counterstain safranin.This lab will employ the Schaeffer-Fu lton Method: there are variations of the spore stainmethod.The identification of spores is also very important for the clinical microbiologist who is analyzinga patient's body fluid or tissue since there are not that many spore-forming genera . In fact, thereare two major pathogenic spore-forming genera, Bacillus and Clostridium , together causing anumber of lethal diseases---botulism, gangrene, tetanus, and anthrax, to name a few. Somespore-forming species produce spores only under adv erse environmental condition, while a fewspecies easily produce spores without m uch prodding. OBJECTIVES: Learn to perform the spore stain. Identify spores on a bacterial smear. MATERIALS NEEDED: dye kitdye stain rackhot platepaper towel (cut the size of the slide) Bacillus culture on an TSA plate THE PROCEDURES: 1. Prepare the bacterial smear of Bacillus , air-dry, and heat-fix.2. Put a beaker of water (a metal beaker) on the hot pla te and boil until steam is coming upfrom the water. Then turn the hot plate down so that the water is barely boiling. Page 41 3. Place the wire stain rack over the beaker which now has steam coming up from the boiledwater.4. Cut a small piece of paper towel and place it on top of the s mear on the slide. The towelwill keep the dye from evaporating too quickly, ther eby giving more contact time betweenthe dye and the bacterial walls.5. Flood the smear with the primary dye, malachite green, and leave for 5 minutes. Keep thep aper towel moist with the malachite green. DO NOT let the dye dry on the towel.6 . Remove and discard the small paper towel piece in the trash.7. Wash really WELL with water and move the slide and wire rack from the boiling water tothe regular stain tray to finish up the last step in the procedure.8. Place the smear in th e stain jar or flood the smear with the counterstain dye, safrinin,and leave for 1 minute.9. Wash WELL with water. Blot dry with bibu lous paper. LABORATORY REPORT SHEET QUESTIONS: 1. Draw some spores along with some vegetative cells for comparison.2. Can you f ind any spores still within the vegetative cell?If so, notice where the spore is in the cell, if there is one---terminal, central, subterminal.Why might that in formation be helpful to a microbiologist?3. What is the purpose of the steam in this stain?4. Why does there not have to be a decolorizer in this stain?5. If yo u mistakenly did this stain on an acid-fast Mycobacterium, what color do you thi nk thecells would come out to be? Page 42 THE ACID-FAST STAIN The ability of the bacteria to resist decolorization with ACID alcohol confers a

cid fastness tothe bacterium. Acid-fast bacteria, of which there are very few--the major genus Mycobacterium , have a high concentration of mycolic acid, a lipid, in their walls. Thisdiffer ential stain is very important for diagnoses of leprosy and tuberculosis, caused by 2different species of the genus Mycobacterium (commonly called AFB in clinical situations).The method used in this lab is the Ziehl-Neelsen method: (there are 2 different methods ofAF staining).As in the sp ore stain, steam is used as a gimmick to get the carbol fuschin primary dye to gointo the wall. Once in, it will not come out: But the acid alcohol decolorizer WILL take it outof the nonacid-fast wall since the primary dye does not bind str ongly to the cell wall. Acid-fastbacteria are hot pink or fuchsia. Nonacid-fast bacteria are light blue. OBJECTIVES: Learn to perform the acid-fast stain.Differentiate between acid-fast and nonacid -fast bacteria. MATERIALS NEEDED: cultures of Mycobacterium smegmatis and E. coli dye kit + dye stain rackhot plate and beakerpaper towel (cut the size of the sl ide) THE PROCEDURES: 1. Prepare the bacterial smear, air-dry, and heat-fix. You will make a mixed suspen sion of Mycobacterium and E. coli on one slide. 2. Put a beaker of water (metal beaker) on the hot plate and boil until steam is co ming upfrom the water. Then turn the hot plate down so that the water is barely boiling. 3. Place the wire stain rack over the beaker which now has steam coming up from the water. 4. Cut a small piece of paper towel and place it on top of the smear on the slide. The towelwill keep the dye from evaporating too quickly, thereby giving more con tact time betweenthe dye and the bacterial walls. 5. Flood the smear with the primary dye, carbol fuschin, and leave for 5 minutes. K eep thepaper towel moist with the carbol fuschin. DO NOT let the dye dry on the towel. 6. Remove the piece of paper and discard in the garbage. Wash the slide really WELL . 7. Add the decolorizer acid alcohol (1% HCl + ethanol) and decolorize for 15-20 sec onds. 8. Wash WELL with water. 9. Flood the smear or place the slide into a jar with the counterstain dye, methyle ne blue,and leave for 1 minute.

10. Rinse with water. Blot dry with bibulous paper. Page 43 LABORATORY REPORT SHEET QUESTIONS: 1. What is chemically unique about the Mycobacterium genus that causes it to be acid-fast?2. How is this stain procedure similar to t he spore stain procedure?3. If you gram stain Mycobacterium , although gram +, is never becomes a dark blue-purple but rather stays a light purple. Why?4. What is the color of E. coli when acid-fast stained? Acid-fast or not acid-fast?What was the color of E. coli when gram stained? Page 44 Lab Manual Add To Collection 31.8K Reads 52 Readcasts 11 Embed Views Published by Yasmine Moise TIP Press Ctrl-F to search anywhere in the document. Sections THE GRAM STAIN THE ENDOSPORE STAIN THE ACID-FAST STAIN THE CAPSULE STAIN TRANSFER OF BACTERIA USING ASEPTIC TECHNIQUE PURE CULTURE TECHNIQUES BACTERIAL COLONY MORPHOLOGY COUNTING BACTERIA EVALUATION OF ANTIMICROBIAL CHEMICALS KIRBY-BAUER TEST FOR ANTIBIOTIC SUSCEPTIBILITY IDENTIFICATION OF UNKNOWN BACTERIA OXYGEN REQUIREMENTS & CULTURING ANAEROBIC BACTERIA CARBOHYDRATE UTILIZATION CASEIN HYDROLYSIS GELATIN HYDROLYSIS GENUS STAPHYLOCOCCUS: Isolation and Identification GENUS STREPTOCOCCUS: Isolation and Identification GLOSSARY OF TERMS Info and Rating Category: Uncategorized. Rating: Upload Date: 04/22/2011 Copyright: Attribution Non-commercial Tags: This document has no tags.

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