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SECTION 1 LABORAT OR Y WEE K 3
DIFFERENTIAL STAINING EX ER CIS E 5. GRAM STAIN CAPSULE STAIN FLAGELLA STAIN EX ER CIS E 6. ENDOSPORE STAIN E XE R CIS E 7. PURE CULTURE TECHNIQUE
DIFFERENTIAL STAINING Definition: A process of staining that facilitates differentiation of various structures in a specimen.
Exercise 5. GRA M STA IN
Hans Christian Gram was a Danish bacteriologist. He developed the Gram stain as a means to differentiate pneumococci ( Labor atory exerc ise 19) from Klebsiella pneumonia (Labora tory exerc ise 25) in 1884. It remains one of the most important staining techniques in microbiology today. The Gram stain is often the first test performed in the identification of bacteria. Gram staining is partly based on the differences in the cell walls of Gram positive and Gramnegative organisms. It is best to use younger cells (1 2-24 hours) because older Gram-positive bacteria are subject to break down of the cell wall by enzymes that are produced with age which may result in Gra m var iab le st aining . Gram-positive organisms have a cell wall composed almost entirely of peptidoglycan whereas Gram-negative organisms have a lipid rich outer wall. This fundamental difference allows the Gram stain to work. The crystal violet complex is an insoluble complex that is difficult to remove. When alcohol is used as a decolorizer, it dehydrates the Gram-positive cell wall and traps the complex inside the cell wall. However, alcohol is able to remove the complex from the lipid rich wall of Gram-negative organisms. The now clear Gram-negative organisms are stained with fuchsin or safranin so that the Gram negative and Gram-positive organisms can be distinguished from one another.
Micrococcus luteus Nutrient Agar Slant Escherichia coli Nutrient Agar Slant
Capsules may be absent. GR AM S TAI NI NG TH E SLID E 2a. 2c. In general. After 30 seconds remove the Safranin and gently wash with water 2i. media rich in sugars and low in nitrogen tend to induce capsule formation.PROC EDUR E Step 1. You may save the slide in your slide box for further study or discard the stained smear in the sharps container. Many bacteria produce capsules under the right conditions. Co lor p lat e 3 . thick. Flood the slide with Safranin.1 CAPSULE STAIN Capsules are structures that lay outside of an organism's cell wall and thus are in direct contact with the environment. Step 3. Capsules can serve the following functions: 3-2 . Record your observations as sketches and descriptions. Some capsules are composed of POLYALCOHOLS or AMINO ACID POLYMERS. thin. 2e. 2f. 2g. 2h. Capsule formation is dependent upon the nutrient conditions. After 1 minute remove the Iodine and wash gently with water. well formed or loose. blot dry with paper towel. Capsules play a number of roles in the life of microbes. OBS ER VATI ON Examine the stained smear with low-power and oil-immersion objectives. 2d. Flood the smear with 95% Ethanol. Place the slide on your staining rack and flood the smear with Crystal Violet. Both prokaryotes and eukaryotes form capsules. After 1 minute remove the stain and wash gently with water. PR EP ARE A FI X ED S ME AR Prepare one fixed smear using both assigned cultures. Bacterial capsules are most often composed of long POLYMERS of sugar (or sugar derivatives) known as POLYSACCHARIDES. and let the slide air dry. 2b. Flood the smear with Grams Iodine. (label the bottom of your slide) Mix both cultures in one smear together Step 2. After 15 seconds remove the Ethanol and rinse gently with water. Drain off excess water.
particularly starvation for nitrogen. and high temperature. and subsequently the destruction of the mother cell. Protect the cell from desiccation (drying). Sporulation has important implications for human health as you will see in laboratory exercise 34 and 35. These genes are responsible for the creation of the spore. low humidity. The smear is prepared in the same manner as the negative stain. This technique takes more time and specialized stains than are usually found in introductory microbiology labs. a whole set of vegetative genes are turned off and a new set of sporulation genes is turned on. its properties are probably related to the exclusion of water from the spore. since application of heat may destroy or distort the capsule. Since the flagella are too thin to be seen in ordinary stains. once stained. The capsule stain technique stains around the capsule with a negative acidic stain. 3. The negative stain is air-dried before the basic stain is applied. There are several staining techniques and some commercially available flagella stains. 2. initially part of the mother cell. at the end (terminal). Co lor plat e 3 . Although it is not completely clear why the spore is so resistant to environmental insults. Endospores themselves. and proteins into the spore. They serve as binding or adhesion agents for sticking cells together and/or to a surface such as a rock in flowing stream or a tooth. During the sporulation process. ribosomes. may be viewed under oil immersion as round or oval. 4. Provide a food reserve when certain organic compounds are in excess.e.1. Then a basic stain will colorize the cell itself. 5. Presence and arrangement of the flagella can be used in identification of the bacteria. heat as high as boiling for up to an hour and drying. Protect the cell from phagocytosis (being engulfed by white blood cells). The polysaccharide or polypeptide composition of the capsules makes staining difficult. the packaging of chromosomal DNA. Co lor p la te 3. 3-3 . spores of pathogenic bacteria introduced into a human body can germinate and lead to disease. and the slide is not heat-fixed. Endospores may be located in the middle of the cells (central). A virulence determinant of pathogenic microbes. While spores can last many thousands of years. Sporulation is induced – turned on – under starvation conditions. The capsule appears as a white halo between the cell and the darker background.3 Exercise 6.2 FLAGELLA STAIN The flagella stain allows for the direct observation of flagella. make endospores) to protect cells through times of poor nutrient availability. special stains and techniques need to be used to coat the flagella with enough stain to obtain a visible thickness. E ND OSP ORE STAIN Bacillus and Clostridium are bacterial genera that sporulate (i. they can be reactivated to generate vegetative cells in response to moisture and proper nutrients. The cell’s DNA is tightly packaged in small basic proteins which protect it from damage by oxygen. or between the end and the middle of the cells (subterminal).
FIGURE 3. and radiation. most disinfectants. CULTURE S Bacillus subtilis Nutrient agar slant Micrococcus luteus Nutrient agar slant 3-4 . 2. DNA repair enzymes contained within the endospore are able to repair damaged DNA during germination. The impermeability of the spore coat is thought to be responsible for the endospore's resistance to chemicals. Finally. The heat resistance of endospores is due to a variety of factors: 1. and drying. The cortex may osmotically remove water from the interior of the endospore and the dehydration that results is thought to be very important in the endospore's resistance to heat and radiation. drying. The cortex is a prominent region composed of loosely cros s. chemicals.1 TH E BACTERIA L ENDOSPORE Exosporium Spore coat (cross-linked Keratin) Spore cortex region (peptidoglycan) Endospore Bacterial endospores are resistant to antibiotics. 3. 4. and physical agents such as radiation. Calciu m-d ip icolinat e is abundant within the endospore. This salt of Dipicolinic acid stabilizes the endospore's DNA along with Specialized DNA-binding proteins that protect it from heat. boiling. De sicca t ion .lin ked p ept id iglyca n .
3-5 . 2c. Cool. PR EP ARE A FI X ED S ME AR Prepare two fixed smear on one slide using your assigned species. Place the slide on your staining rack and flood the smear with Malachite Green. (label the bottom of your slide) Step 2. 2b. Drain off excess water. After 30 seconds remove the Safranin and gently wash with water 2e. Stea m s lide for 3-5 minu tes (t he in struc tor will de mon s trat e). OBS ER VATI ON Examine the stained smear with low-power and oil-immersion objectives. Record your observations as sketches and descriptions.PROC EDUR E Step 1. and let the slide air dry. blot dry with paper towel. remove the stain and wash gently with water. E ND OSP ORE S TAI NI NG TH E S LIDE 2a.4 You may save the slide in your slide box for further study or discard the stained smear in the sharps container. Co lor pla te 3. 2d. Step 3. Flood the slide with Safranin.
luteus Yellow colonies. smooth. and isolated from. irregular M. PURE CULTUR E TECHNIQUE The skin and many mucosal surfaces of the human body support large numbers of microorganisms that comprise the normal. other harmless organisms. smooth. small round. small round. The mixture contains the following microorganisms: E. (slow growth) Streak Mixed Broth for Isolation (the instructor will demonstrate this procedure) 3-6 . flat. CE LL GR OUPI NG AND S TAI NI NG RE ACTI ON 10 00X Escherichia coli Micrococcus luteus AS SI G NE D MI CR OOR G ANIS M E ND OS P OR E STAI NI NG D ATA DI AG R AM: MORP H OLOG Y.1 AS SI G NE D MI CR OOR G ANIS M GR AM S TAI NI NG D ATA DI AG R AM: MORP H OLOG Y. flora. CULTURE S Each desk is provided with a mixed broth culture labeled “Mixed Bacteria”. coli White colonies. accurate identification of microorganisms. CE LL GR OUPI NG AND S TAI NI NG RE ACTI ON 10 00X Bacillus subtilis Micrococcus luteus Exercise 7. Colonies of the pathogenic species must be picked out of the mixed culture and grown in isolated pure culture. The microbiologist can then proceed to identify the isolated organism by examining its biochemical and immunological properties ( la boratory w eek 6 a nd 13) . or indigenous. any pathogenic microorganisms being sought must be recognized among. convex. Pure culture technique is critical to successful. roseus Red colonies. When clinical specimens are collected from these surfaces and cultured. convex M.RESULTS TABLE 3.
Flame loop. Step 4. This allows for individual colonies to be isolated from other colonies. Flame the loop. Now rotate the plate. rotate plate. The proper wrist action and light touch takes practice. Each colony is considered "pure. quadrant of the agar plate (See f igur e 3. cool by touching an uninoculated portion of the surface. Open lid and streak again. Step 1. following the diagram Remember: you are picking up growth from quadrant one. Incubate plate inverted at room temperature until the next laboratory meeting. and using this as your inoculum for quadrant two. FIGURE 3. Comments and suggestions: 3-7 .2 STREAK FOR ISOLATI ON Culture flame loop flame loop flame loop Quadrant 1 Quadrant 2 Quadrant 3 Quadrant 4 RESULTS Draw your results showing the approximate number of isolated colonies and record any suggestions the instructor may have for improvement. Don't penetrate or scrape the agar surface.PROC EDUR E The streak plate technique is essentially a method to dilute the number of organisms. Cover plate with lid. decreasing the density. Use a light touch. Begin with inoculating the first." since theoretically. Step 3. Step 5. and repeat procedure for quadrants three and four.2 b elo w) . Step 2. the colony began with an individual cell. or primary.
What is the function of the Iodine solution in the Gram stain? 2.TERMS AND QUEST IONS F OR STUDY 1. Describe two conditions in which an organism might stain gram variable? 6. Describe the process of endosporulation. What is the value of a capsule to a microorganism? 3-8 . the flagella stain and the endospore stain? 8. Why is the location of an endospore important? 10. What is the function of the 95% ethanol in the Gram stain? 3. What counter stain is used in the Gram stain and why is it necessary. 4. What is the accepted theory regarding the mechanism of the Gram stain? 7. What is the importance of the capsule stain. is this a reproductive mechanism? Explain. 9. What is the advantage of the Gram stain over the simple stain? 5.
When an agar plate is inoculated. luteus ? 15. What is a mixed culture? What is a pure culture? 13. coli from M. Why is it necessary to isolate individual colonies from a mixture in the clinical lab? 3-9 .11. What is a bacterial colony? 14. How would you differentiate colonies of E. why is the loop flamed between quadrants? 12.