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LAB 3Bacterial Staining Techniques III. Differential Stains: Gram Stain and Acid-fast StainII. Morphological UnknownI. DIFFERENTIAL STAINSA. Gram StainB. Acid-fast StainA. Gram StainThe previous lab introduced simple staining techniques that enable microbiologists to observe themorphological characteristics of bacteria. Although simple stains are useful, they do not reveal detailsabout the bacteria other than morphology and arrangement. The Gram stain is a differential staincommonly used in the microbiology laboratory that differentiates bacteria on the basis of their cell wallstructure. Most bacteria can be divided into two groups based on the composition of their cell wall:1) Gram-positive cell walls have a thick peptidoglycan layer beyond the plasma membrane.Characteristic polymers called teichoic and lipoteichoic acids stick out above the peptidoglycanand it is because of their negative charge that the cell wall is overall negative. These acids arealso very important in the body’s ability to recognize foreign bacteria. Gram-positive cell wallsstain blue/purple with the Gram stain.2) Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an outermembrane beyond the plasma membrane. The space between the plasma membrane and the outermembrane is called the periplasmic space. The outer leaflet of the outer membrane is composedlargely of a molecule called lipopolysaccharide (LPS). LPS is an endotoxin that is important intriggering the body’s immune response and contributing to the overall negative charge of the cell.Spanning the outer membrane are porin proteins that enable the passage of small molecules.Lipoproteins join the outer membrane and the thin peptidoglycan layer. Gram-negative cells willstain pink with the Gram stain.This is The Most Important staining technique in Bacteriology.Cell wall structure of Gram and Gram-21

GRAM STAINProcedureFixed cells on slidePrimary stainMordantDecolorizerCounterstainReagentCrystal VioletIodineAlcoholSafraninCell ColorGram PositiveCOLORLESSPURPLEPURPLEPURPLEPURPLEGram NegativeCOLORLESSPURPLEPURPLECOLORLESSREDAn easy way to remember the steps of the Gram stain is.22

PROCEDURE: (EACH STUDENT)1. Using a sterile inoculating loop, add 1 drop of sterile water to the slide. Prepare a mixed smear ofEscherichia coli (G- rod) and Staphylococcus epidermidis (G coccus).2. Air dry and Heat fix.3. Cover the smear with Crystal Violet (primary stain) for 1 min.4. Gently wash off the slide with water.5. Add Gram’s Iodine (mordant) for 1 min.6. Wash with water.7. Decolorize with 95% ethanol. This is the "tricky" step. Stop decolorizing with alcohol as soon as thepurple color has stopped leaching off the slide (time will vary depending on thickness of smear).Immediately wash with water. Be sure to dispose of all ethanol waste in the appropriately labeled wastecontainer.8. Cover the smear with Safranin for 30 seconds.9. Wash both the top & the bottom of the slide with water.10. Blot the slide with bibulous paper.11. Using the 10X objective lens, focus first on the line and then on the smear. Follow the focusingprocedure in Lab #1. Use the focusing procedure in Lab #2 to view the smear using the 100X (oilimmersion lens).Focus lineE. coliS. epidermidisNote: Escherichia coli is a tiny pink (Gram-) rod. Staphylococcus epidermidis is a purple (Gram ) sphereor coccus.Draw a picture of a typical microscopic field and identify both Escherichia coli and Staphylococcusepidermidis. Record this in the results section for this lab. Colored pencils are available throughout the roomon the chalkboard trays.23

B. Acid-fast StainMycobacterium and many Nocardia species are called acid-fast because during an acid-fast stainingprocedure they retain the primary dye carbol fuchsin despite decolorization with the powerful solventacid-alcohol (95% ethanol with 3% HCl). Nearly all other genera of bacteria are nonacid-fast. The acidfast genera have the waxy hydroxy-lipid called mycolic acid in their cell walls. It is assumed thatmycolic acid prevents acid-alcohol from decolorizing protoplasm. The acid-fast stain is a differentialstain.Ziehl Neelsen Acid-fast stainACID-FAST STAINProcedurePrimary -alcoholMethylene blueCell ColorAcid-fast BacteriaNonacid-fast BacteriaREDREDREDCOLORLESSREDBLUEPROCEDURE - (EACH STUDENT)1. Add one loopful of sterile water to a microscope slide.2. Make a heavy smear of Mycobacterium smegmatis. Mix thoroughly with your loop. Then transfer asmall amount of Staphylococcus epidermidis to the same drop of water.You will now have a mixture of M. smegmatis and S. epidermidis.3. Air dry and heat fix well.4. Cover the smear with carbolfuchsin dye. Place a piece of paper towel on top of the dye. Be sure thepaper towel is saturated with the dye. Carbolfuchsin is a potential carcinogen. Please wear gloves whenworking with this dye.5. Place the slide on the rack over dry heat for 2 minutes.6. Cool and rinse with water.7. Decolorize by placing a drop of acid alcohol on the slide and allowing it to sit for 15 seconds.8. Wash the top and bottom of slide with water and clean the slide bottom well.9. Counterstain with Methylene Blue for 30 seconds to 1 minute.10. Wash and blot the slide with bibulous paper.11. Focus 10X - then use oil immersion.M. smegmatis (heavy)Focus lineS . epidermidisDraw a typical microscopic field and record in Results Lab 3.Note: The acid-fast Mycobacterium retains carbolfuchsin and stains hot pink. The Staphylococcusepidermidis is decolorized and the counterstain colors them blue. See table for acid-fast stain.24

II.1.2.3.Morphological Unknown: continue your investigation of your morphological unknown.Collect your unknown from the side bench. Verify that the # matches that used for your direct stain.Perform a Gram stain and an Acid-fast stain as described in the above sections.Record your observations in the Results section of Lab #4.25

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LAB 3 RESULTSI. DIFFERENTIAL STAINSA. Gram Stain Draw and label examples of Escherichia coli and Staphylococcus epidermidis.B. Acid-fast Stain Draw and label examples of Mycobacterium smegmatis and Staphylococcus pidermidisQUESTIONS:1. What is the difference between a simple and a differential stain?2. Describe the function of each of the following in the Gram stainMordant:Primary stain:Decolorizer:Counterstain:3. Which step in the Gram stain is most likely to cause poor results if done incorrectly?4. Why must fresh bacterial cultures be used in a Gram stain?27

5. Briefly describe the mechanism of Gram staining.6. What is the primary stain used in the acid-fast staining procedure?7. What is the purpose of the heat/steam during the acid-fast staining procedure?8. In a clinical microbiology laboratory, the acid-fast stain would be used for diagnosis of what diseases?9. What makes a microorganism nonacid-fast?Organisms introduced in this lab:Mycobacterium smegmatisMycobacterium tuberculosisMycobacterium lepraeStaphylococcus epidermidis28

GENERAL MICROBIOLOGY 2210: GRAM STAIN REPORT (15 PTS)ASSIGNMENTEach student will have the opportunity to submit one example of a Gram stain with a brief written reportdescribing the staining procedure and the stain itself. A good report will likely necessitate a minimum of 2pages double-spaced and is limited to a 3-page maximum.OVERALL FORMATPlease include the following in your report:A. Title Page with the following information: (1 point)--Title: A title should be brief, creative, but descriptive of the work that was done.--Your Name--Course and Lab Section--DateB. Introduction: (4 points)An introduction should include pertinent background information. In this case, the history of the Gramstain and its significance in microbiology are relevant (1 point). A discussion of the mechanism of Gramstaining and how it differentiates bacteria on the basis of their cell wall structure should also be included(1 point). The purpose and objectives of the experiment should be stated and a hypothesis should bemade (2 points). Remember that a hypothesis need not be correct, but it must be tested by the procedure.Good hypotheses are also grounded in background knowledge and clearly state the predicted results.Hypotheses can be of several types. 1 We will discuss two types here:1. Manipulated hypothesesThis type of hypothesis is used when one variable (the independent variable) is manipulated by theexperimenter and the effects on a second variable (the dependent variable) are observed. This type ofhypothesis is generally written as an if, then statement (e.g. If the smear of the unknown bacterium isdecolorized as according to the above procedure (neither over- nor under-decolorized), then all thecells will stain a single color.) In this example, decolorization is the independent variable (thevariable controlled by the experimenter) and the color of the cells is the dependent variable (thevariable affected by the changes in the independent variable).2. Observational hypothesesStates something about the nature of an organism (e.g. The unknown bacterium will stain Grampositive.)C. Materials and Methods: (4 points)The Materials and Methods section should include a description of the procedure/steps in the Gram stain,including the preparation of a bacterial smear and heat fixation (3 points). This section must be writtenin complete sentences and in paragraph form (1 point). Illustrations may assist in clarifying points.D. Results: (1 point)In the results section, describe your Gram stain/s. Include at least one illustration of your owncreation.E. Discussion: (3 points)What features of your Gram stain exemplify the Gram stain theory (1 point)? If there are features of theGram stain that are not as expected, what possible sources or error may be responsible (e.g. poor qualityof the smear preparation)? (1 point) Discuss whether the hypothesis made in the introduction was testedand if so was it supported? (1 point)1For a discussion of hypotheses formation please HF2C.HTM29

OVERALL QUALITYStudents have the opportunity to earn points for reports that are well written, organized and edited.(2 points)DUE DATE: TAs will inform you of the due date.*Please tear this grading key out of your lab book and attach it to your lab report at the time ofsubmission. Please also write your name and lab section on the frosted portion of your Gram stainslide and hand it in with your written report.TA comments:TA’s suggestions for improving Gram stain:Points earned out of 15 possible:30