Why should the smear be rinsed with tap water?

Why should the smear be rinsed with tap water?

You want to wash your slide before blocking them to get rid out of the acid and not over-digesting. Tap water has a nice turbulence which make a very efficient washing step.

Why do you rinse the slide with water after staining?

The decolorization of the cells is the most “operator-dependent” step of the process and the one that is most likely to be performed incorrectly. Rinse with water to stop decolorization. Rinse the slide with a counterstain (safranin or carbol fuchsin) which stains all cells red.

Will you rinse off the glass slide with water or continue without washing?

Overheating the glass slide may create microfissures in the glass that will retain stain and produce inconclusive results. It doesn’t really matter. It fixes the cells to the slide to prevent cells from washing off during the washing step. It differentiates gram positive from gram negative cells.

What is the safest way of drying bacterial smear?

In order to heat fix a bacterial smear, it is necessary to first let the bacterial sample air dry. Then either place the slide in the slide holder of a microincinerator, or pass the dried slide through the flame of a Bunsen burner 3 or 4 times, smear side facing up.

Why must a smear be air dried before heat fixing?

Air drying the smears before heat fixation is essential. Water can boil while passing the slide through the flame which could alter the natural shape and size of the bacteria. Overheating the smear during heat-fixing process can distort the form and structure of the cells.

Why must slides be clean grease free?

After removing the soapy filling with a clean cloth the surface becomes clean and grease free. If the slide is perfectly clean drop can spread on its surface in a thin even film, otherwise the water collects into small drops and film cannot be made.

What happens if you put too much bacteria on a slide?

If your slide is wet and you heat fix it, the bacteria will boil and the cellular morphology will be lost. If your slide is wet and fix it in methanol, it will most likely wash off the slide. Smears that are too thick will most likely wash off the slide regardless of the fixation method.

Is a positive control properly stained?

Positive control: Staphylococcus aureus (should stain blue to dark purple/gram-positive cocci) Negative control: Escherichia coli (should stain pink to red/gram-negative rods)

What happens if you overheat a bacterial smear?

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.

Why is it important to use a small amount of bacteria when making a smear?

Just as in preparing a smear, you only need a small amount of organism. If you have too many organisms, you won’t be able to see the morphology of individual cells. It is also important not use too much nigrosin. If it is too thick, the background will have a cracked appearance similar to mud puddles drying in the sun.

What is it important to prepare a good bacterial smear?

Before staining and observing a microbe under a microscope, a smear must be prepared. The goal of smear preparation is to place an appropriate concentration of cells on a slide and then cement them there so that they do not wash off during the subsequent staining procedure.

Why can’t you heat fix the smear until the smear is completely dries?

Why must a smear be dry before heat fixing? Air drying the smears before heat fixation is essential. Water can boil while passing the slide through the flame which could alter the natural shape and size of the bacteria. Overheating the smear during heat-fixing process can distort the form and structure of the cells.

What’s the best way to fix a bacterial smear?

There are two methods of adhering your bacteria to the slide, heat fixation or methanol fixation. Heat fixing is only used with BSL1 organisms. The organisms we will be working with are BSL2, so you will need to use the methanol fixation technique.

How does a smear for a Gram stain work?

Smears typically require only a small amount of bacterial culture. An effective smear appears as a thin whitish layer or film after heat-fixing. 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. Allow the smear to air dry.

Do you need a small amount of organism to prepare a smear?

Just as in preparing a smear, you only need a small amount of organism. If you have too many organisms, you won’t be able to see the morphology of individual cells.

What’s the best way to make a Gram smear?

Wipe the slides with spirit or alcohol. After cleaning, dry the slides and place them on laboratory towels until ready for use. Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to clearly designate the area in which you will prepare the smear.