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Micro Lab 5
| Question | Answer |
|---|---|
| Purpose of Staining Experiment | The purpose of this experiment was to perform a negative stain and gram stains on different specimens. |
| Specimens Used | Unknown, Bacillus subtilis (negative stain), Micrococcus luteus (gram positive control), Pseudomonas flourescens (gram negative control) |
| Micrococcus luteus | Cocci, purple cells. The purple color led to the identification of Micrococcus luteus as gram positive, meaning it has a thick peptidoglycan wall and retained the crystal-violet better. |
| Pseudomonas flourescens | Bacillus, pink cells. The pink color led to the identifcation of Pseudomonas flourescens as gram negative, meaning it has a thin peptidoglycan wall and was more susceptible to the counterstain. |
| Bacillus subtilis | The negative stain experiment was successful, because it correctly showcased the shape and structure of the cells without any of the additional details found in gram stains. Colorless, rod-shaped |
| Common Cell Types | The gram positive characteristic is unique to some bacteria, yeasts, and a few molds. Most other living cells are gram negative or gram variable. A few other are gram non-reactive, like Mycobacterium. |
| Gram-positive bacteria | the cell wall of Gram-positive bacteria has peptidoglycan, a polysaccharide with peptide cross-linkages. The crystal violet can't pass through peptidoglycan during decolorization, as the solvent causes the thick peptidoglycan to contract and thus retain. |
| Gram-negative bacteria | peptidoglycan is a minor component and there is also a proteolipid, neither of which prevents the stain loss. If the thick peptidoglycan is removed from a gram-positive cell, the cells will not retain the stain complex and will appear Gram-negative. |
| General Protocol for Gram Stain | 1) Primary stain 2) Mordant 3) Decolorizing agent 4) Counterstain |
| Primary Stain | crystal violet, stains all cells purple. |
| Mordant | enhance a formation between dye and the bacterial cell. An iodine solution serves is the mordant. |
| Decolorizing agent | acetone, alcohol, etc. Removes primary stain from Gram-negative and "dehydrates" the Gram-positive. |
| Counterstain | recolors cells that have lost the primary stain after alcohol treatment |
| Why is decolorization a critical step in the procedure? | Decolorization is a critical step in procedures like Gram staining because it differentiates between cellular components, such as bacteria, by selectively removing a primary stain from some while leaving it on others. |
| What did we used as a decolorizer? | Decolorizer: Acetone-Ethanol (50:50 - Red squirt bottle) |
| Example of Gram-Negatives | Enterobacter aerogenes Escherichia coli Pseudomonas fluorescens Citrobacter freundii |
| Example of Gram-Positives | Bacillus subtilis Staphylococcus epidermidis Micrococcus luteus Enterococcus durans |
| Gram Stain Procedure | Prepare three smears, then heat-fix them. Stain the slide with crystal violet, rinse with water, apply iodine, rinse again, then decolorize until no purple runoff. Rinse immediately with water, counterstain with safranin for 2 minutes, rinse and blot dry. |
| Why do we use a wax pencil? | The wax is water-resistant, so it doesn't wash off during staining. |
| Heat Fixing | To heat fix, first let the smear air-dry completely. Then, pass the slide quickly through the flame of a Bunsen burner in a P shape. We heat fix to kill the bacteria and preserve their shape so they don’t wash off or distort during staining. |
| Brightfield Microscopy with Gram Stains | Examine the slides under bright-field with no cover slip. Focus the slide beginning at 100x total magnification (10x objective). You will need the oil-immersion objective (1000X) to judge the quality of your Gram stain. |
| Negative Stain | determines cellular morphology and to view capsules. Since the cells themselves are not stained, their morphology is not distorted in any way. The nigrosin provides a dark background against which the shapes of the unstained cells are clearly visible. |
| Negative Stain Procedure | Place a drop of nigrosin on a slide, mix in a small amount of organism, and use another slide to spread the mixture into a thin film. Let it air dry completely, then observe under microscope. |
| Gram Stain Vs Negative Stain | Gram stain: Differentiates bacteria into two major groups (Gram-positive and Gram-negative) based on their cell wall structure Negative stain: Determines cellular morphology and allows visualization of capsules without distorting the cells. No colors. |
| After correct Gram staining, the gram-negative cells will appear _____, whereas the gram-positive cells appear _____ | pink purple |
| Your Gram stain is complete and correct. Which of the following statements would apply to the image you see? Select all that apply | you can observe: -gram-neg bacilli -& gram-pos cocci |
| After you complete the staining, you observe only gram negative cells. Which of the following statements would apply to the image you see? | This image could be explained by omission of the Gram's iodine step, causing all cells to appear gram-negative. the primary crystal violet stain won't be fixed to the cell walls and will be washed away during the decolorization step. |
| After you complete the staining, you realize that you did not perform the safranin step. Which of the following statements would correctly describe what you would see? | Gram-negative organisms might not be visible. Gram pos would be purple and Gram neg would be colorless. |
| After you complete the staining, you realize that you performed the decolorizer step for 10 minutes instead of 10 seconds! Which of the following statements would correctly describe what you would see? | Gram-positive organisms might incorrectly stain pink. Both gram pos and gram neg would be stained pink. |
| After you complete the staining, you realize that you did not use the alcohol bottle at all. Which cells would appear purple? Select all that apply. | Gram-negative cells Gram-positive cells |
| Why do all my cells appear pink, including the Gram-positive control? | You over-decolorized by leaving the decolorizer on too long or using a smear that was too thin. |
| Why do all my cells appear purple, including the Gram-negative control? | You under-decolorized by not applying the decolorizer long enough or the smear was too thick. |
| Why do my Gram-positive cells appear pink even though I followed all the steps? | You likely skipped or didn't apply the iodine mordant for the full minute. |
| Why does the same organism show both purple and pink cells on my slide? | Your culture is too old (over 24 hours) or you have a contaminated mixed culture. |
| Why are my cells barely visible and very faintly colored? | Your crystal violet or safranin reagents are too old, too dilute, or you didn't stain long enough. |
| Why can't I see any cells on my slide at all? | You didn't heat-fix the slide properly, so the smear washed off during staining. |
| Why are my bacteria clumped together instead of individual cells? | You used too much culture material or didn't mix and spread it properly with water. |
| Why is my slide background stained purple instead of clear? | You didn't wash off excess crystal violet thoroughly enough after the primary stain step. |
| Why are my known control organisms showing the wrong Gram reaction? | Your control cultures are contaminated, too old, or your reagents have expired. |
| How long should I decolorize to avoid both over and under-decolorization? | Decolorize until the purple color just stops draining from the slide into the waste container. |
| What happens if I forget to blot dry after washing off the iodine? | Excess water will dilute the decolorizer, causing under-decolorization and false Gram-positive results. |
| Why do my results vary every time I stain the same organism? | You're inconsistent with timing, especially during the critical decolorization step. |
| What causes Gram-positive bacteria to lose their ability to retain crystal violet? | Old cultures, damage to cell wall |
| Why is it critical to use cultures that are 18-24 hours old? | Older Gram-positive cultures may have damaged cell walls and appear Gram-negative. |
| What's the most common mistake that causes false Gram-negative results? | Over-decolorization, which strips the crystal violet-iodine complex from Gram-positive cells. |
| What's the most common mistake that causes false Gram-positive results? | Under-decolorization, which leaves the crystal violet-iodine complex in Gram-negative cells. |
Created by:
smurtab