How do you fix a 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. Once the slide is heat fixed, it can then be stained.

Then, what are the other ways of fixing the bacteria to the slide?

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.

Similarly, why would you heat fix a bacterial smear? Before bacteria can be stained, a smear of bacteria must be made on a slide and heat fixed. Heat fixing denatures bacterial enzymes, preventing them from digesting cell parts, which causes the cell to break, a process called autolysis. The heat also enhances the adherence of bacterial cells to the slide.

In this regard, how do you make a bacterial smear?

SMEAR PREPARATION

Place one needle of solid bacterial growth or two loops. of liquid bacterial growth in the center of a clean slide.
If working from a solid medium, add one drop (and only one drop)
Now, with your inoculating loop, mix the specimen with the water.
Place the slide on a slide warmer and wait for it to dry.

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.

35 Related Question Answers Found

What are the types of fixation?

Types of fixation

Physical methods include heating, micro-waving and cryo-preservation (freeze drying). Heat fixation is rarely used on tissue specimens, its application being confined to smears of micro organisms.

Does heat fixing kill 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.

What is a simple stain?

The simple stain can be used to determine cell shape, size, and arrangement. True to its name, the simple stain is a very simple staining procedure involving only one stain. Basic stains, such as methylene blue, Gram safranin, or Gram crystal violet are useful for staining most bacteria.

What advantages are there to observing unstained bacterial specimens?

Advantages. Brightfield microscopy is very simple to use with fewer adjustments needed to be made to view specimens. Some specimens can be viewed without staining and the optics used in the brightfield technique don't alter the color of the specimen.

Why do we use mordant?

A mordant or dye fixative is a substance used to set (i.e. bind) dyes on fabrics by forming a coordination complex with the dye, which then attaches to the fabric (or tissue). In the past, it was thought that a mordant helped the dye bite onto the fiber so that it would hold fast during washing.

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

A bacterial smear is simply that—a small amount of culture spread in a very thin film on the surface of the slide. To prevent the bacteria from washing away during the staining steps, the smear may be chemically or physically “fixed” to the surface of the slide.

How would you prepare a permanent slide of bacteria?

To prepare the slide:

Place a drop of fluid in the center of the slide.
Position sample on liquid, using tweezers.
At an angle, place one side of the cover slip against the slide making contact with outer edge of the liquid drop.
Lower the cover slowly, avoiding air bubbles.
Remove excess water with the paper towel.

How do you make a perfect smear?

Place clean glass slide on a flat surface. Add one small drop of blood to one end.
Take another clean slide, and holding at an angle of about 45 deg, touch the blood with one end of the slide so the blood runs along the edge of the slide by capillary action.
Make 2 smears, allow to air dry, and label clearly.

What is smear fixation?

Heat fixation: After a smear has dried at room temperature, the slide is gripped by tongs or a clothespin and passed through the flame of a Bunsen burner several times to heat-kill and adhere the organism to the slide. Routinely used with bacteria and archaea. Perfusion: Fixation via blood flow.

Why mycobacteria are acid fast?

These Acid-fast organisms like Mycobacterium contain large amounts of lipid substances within their cell walls called mycolic acids. These acids resist staining by ordinary methods such as a Gram stain. It can also be used to stain a few other bacteria, such as Nocardia. Acid-fast bacilli are bright red after staining.

What is a smear to a microbiologist?

Microbiology - 003 - Bacterial Smear and Simple Stain. Making a bacterial smear prepares the bacteria to be stained and is the first step of most staining procedures. A successful smear will have a single layer of bacteria fixed to the slide, ready to be stained and then observed under a microscope.

What is a stain in microbiology?

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Biological staining is also used to mark cells in flow cytometry, and to flag proteins or nucleic acids in gel electrophoresis.

How long does it take to sterilize an inoculating loop or needle?

Replace the cap on the tube and place it on the rack. Sterilize the loop by flaming it. Now you may lay it down on the lab bench or return it to its container. Incubate the culture you just inoculated at 37°C for 24-48 hours.

How does a Gram stain work?

The Gram stain procedure distinguishes between Gram positive and Gram negative groups by coloring these cells red or violet. Gram positive bacteria stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with.

What other methods of fixation can be used for smear preparation?

2 The commonly used methods are air-dried and wet-fixed smears. Air- dried smears have many advantages over wet-fixed smears during routine cytology. They may be post- fixed after rehydration in saline with a variety of fixatives, such as ethanol/acetic acid, 95% ethanol or alcoholic formalin.

Why do we stain organisms?

The most basic reason that cells are stained is to enhance visualization of the cell or certain cellular components under a microscope. Cells may also be stained to highlight metabolic processes or to differentiate between live and dead cells in a sample.

What is the purpose for making a smear during negative staining?

Negative Stain. In contrast to direct stains that bind to bacteria directly, a negative stain colors the background of a smear rather than the bacteria. These stains have negatively charged functional groups so they can't bind directly to negatively charged bacteria.