What happens if you stain bacteria for too long?
What happens if you Decolorize a Gram stain too long quizlet? If you leave the decolorizer on too long: gram positive bacteria will come out pink. If you don't leave it on long enough: gram negative bacteria will come out purple.
What will happen if the bacterial smear on the glass slide is heated for too long?
If the smear is overheated during heat fixing, the cell walls will rupture. Concentration and freshness of reagents may affect the quality of the stain. Washing and drying of the smear between steps should be consistent. Excess water left on the slide will dilute reagents, particularly Gram's iodine.
What are two common mistakes when making a bacterial smear for staining?
Match
- Applying too much bacteria to the slide.
- Not heat fixing the bacteria before staining.
- Wiping off the slides will result in a loss of bacteria present.
What happens if Decolorizer is left on too long in Gram stain?
The decolorizer should stay on the slide for no more than 15 seconds! If the decolorizer is left on too long, even gram positive cells will lose the crystal violet and will stain red. The staining procedure is here.
What are the common errors in staining methods?
The rank of errors is:
- Over decolourisation.
- Mixed cultures.
- Misread stains.
- Aged subcultures.
- Disorganisation.
- Inadequate fixation and Insufficient culture.
Sep 27, 2020
Why is time an important factor in simple staining?
Why is time an important factor in simple staining? Time is important because it creates a contrast between the bacteria and the stain. If you over or under stain you won't be able to see bacteria.
Why is it important to fix bacterial smears on a slide?
The purpose of making a smear is to fix the bacteria onto the slide. Fixing the bacteria will preserve the morphology of the cells long-term. Also, fixation assists the cells in adhering to the slide, so that the cells do not fall off the slide during the staining procedure.
Why is an 18 24 hour culture necessary for a Gram stain?
Old cultures or smears could give atypical results. That is why cultures of 18-24 hours or recent smears are recommended. It is very important to control the heat-fixation (few seconds), any excess heating could produce erroneous results. Highly chlorinated tap water could weak the counter staining.
What would you observe if you Decolorized your slide too much?
What would you observe if you decolorized your slide too much? How would your cells appear? All of your cells would appear pink if you decolorized your cells too much with acetone-alcohol. Acetone-alcohol, if used too liberally, will wash the crystal violet out of the cells in your smear.
What is the purpose of fixing the smear that is to be stained?
This process is called heat fixing the specimen to the slide. Its purpose is to bind the specimen to the slide so that it does not wash off during staining. Killing the cells with heat fixation also increases their permeability to the dyes used in staining.
Why is using cultures older than 24 hours not advisable in Gram staining method?
Old cultures tend to lose the peptidoglycan cell walls, which predisposes gram-positive cells to be gram-negative or gram variable.
Why is it important to limit the quantity of cells on a smear?
why is it important to limit the quantity of cells used to prepare a smear? large amounts of cells in a smear can cause staining artifacts because stain is not washed away by destaining agents or water.
What effects will improper fixing have on the smear?
Killing the cells with heat fixation also increases their permeability to the dyes used in staining. Do not under-fix (the smear will wash off) nor over-heat (the cells will be ruptured or distorted) your specimen. The slide should be warm to the touch, not hot.
What are the potential consequences of making a smear that is too thick?
Insufficiently dried smears (and/or smears that are too thick) can detach from the slides during staining. The risk is increased in smears made with anticoagulated blood. At room temperature, drying can take several hours; 30 minutes is the minimum; in the latter case, handle the smear very delicately during staining.
What causes a stain to adhere to bacterial cells?
Basic: A stain that has positively charged ions (cationic) that will react with material that is negatively charged. The surface of bacteria is somewhat negatively charged, therefore basic dyes will permeate the cell wall and will be attracted to negative charges of the bacteria and stain the bacteria.
What are the common errors in staining procedure?
The rank of errors is:
- Over decolourisation.
- Mixed cultures.
- Misread stains.
- Aged subcultures.
- Disorganisation.
- Inadequate fixation and Insufficient culture.
Sep 27, 2020
How can the thickness of the smear affect the staining procedure?
How can the thickness of the smear affect the staining procedure? If it is too thick it won't stain properly. The stain will not get to the lower layers and clumps of cells may not be decolorized.
Why is it important not to overheat a bacterial smear?
Why should care be exercised not to overheat a bacterial smear when heat fixing to a slide? Overheating the slide can cause gas to expand in the slide and shatter it, presenting a physical danger. It will damage the cell wall and could lead to inconclusive results due to poor stain retention.
What happens if Safranin is left on too long?
The gram-negative cells will lose color, while the gram-positive cells will remain violet or blue. However, if the decolorizer is left on too long, all cells will lose color! Apply the secondary stain, safranin, and allow it to sit for 1 minute. Gently rinse with water no longer than 5 seconds.
What might happen if a smear is too thick?
Smears that are too thick will be difficult to decolorize and imposable to read. Do not cover the entire slide with the sample. This will make handling difficult and areas may be missed during decolorization. An area the size of a nickel usually is adequate.
What might happen if you heat your slide longer than 2 seconds?
Overheating the slide can cause gas to expand in the slide and shatter it, presenting a physical danger. It will damage the cell wall and could lead to inconclusive results due to poor stain retention.
What would probably happen if you forgot to heat fix the smear before staining?
Heat-fixing the slide fixes the bacteria to the slide surface. If this step is not done, the bacteria in the smear would be washed off of the slide during the staining and decolorization steps. You would not see anything on the slide under the microscope.
What would happen if you left the alcohol on too long during the Gram stain?
Over-decolorization: Leaving alcohol (decolorizer) on the slide for too long disrupts the cell wall of both Gram-positive and Gram-negative bacteria. The Gram-positive bacteria can not retain Crystal Violet, and therefore, all bacteria appear Gram-negative (pink) after counterstaining with Safranin.
Which of the following happens if the bacterial smear is applied in too thick of a layer?
too thick? In a thick smear the bacteria will be too concentrated, reducing the amount of light passing through the slide, the stain may not penetrate adequately, and it will be difficult to visualize individual cells.
What would happen if students forgot to heat fix their bacterial smears slides before performing a Gram’s stain?
Heat-fixing the slide fixes the bacteria to the slide surface. If this step is not done, the bacteria in the smear would be washed off of the slide during the staining and decolorization steps. You would not see anything on the slide under the microscope.
What if the smear you prepare becomes too dense?
2. What if the smear you prepare becomes too dense? When the smear becomes too dense it becomes harder for light to pass through which makes it har to see the shape and size of the organism.
What would happen if you forgot to heat fix a smear prior to staining?
Heat-fixing the slide fixes the bacteria to the slide surface. If this step is not done, the bacteria in the smear would be washed off of the slide during the staining and decolorization steps. You would not see anything on the slide under the microscope.
Why is it important to heat fix the slide before staining?
Heat fixation ensures the elimination of contaminating organisms from the smear preparation. Heat fixation adheres the cells to the slide and coagulates the bacterial proteins, effectively killing the bacteria. Heat fixation is necessary to provide contrast between the cells and the background.
Why is it important to heat fixing the bacteria?
Heat fixing kills the bacteria in the smear, firmly adheres the smear to the slide, and allows the sample to more readily take up stains.
Why is it important to heat fix a bacterial smear before staining?
Heat fixation ensures the elimination of contaminating organisms from the smear preparation. Heat fixation adheres the cells to the slide and coagulates the bacterial proteins, effectively killing the bacteria. Heat fixation is necessary to provide contrast between the cells and the background.