por What is the bacterial smear?
He bacterial smear It is an extension in the form of a thin film of a suspension of bacterial microorganisms that is carried out on a transparent glass plate or slide, for its observation under an optical microscope.
The extension in the form of a film is made in order to separate the microorganisms as much as possible, since if they are grouped the observation is not clear.
In the study of bacterial cultures, smear preparation, fixation and staining techniques are used to better analyze them. Due to the small size of microorganisms, the use of an optical microscope is necessarily required for their observation.
Light microscopes are indispensable instruments for the observation of smears. These employ optical lenses and light, allowing the visualization of samples at high magnification.
In general, living cells do not have mostly colored structures, under the light microscope they are colorless, transparent samples and show very little internal contrast and with their environment.
Observation with a simple bright field light microscope, without the use of auxiliary staining techniques, is very limited and is only used in some cases, such as in the observation of the movement of microorganisms.
For optimal observation of microorganisms, a balance between contrast and resolution must be achieved. Cell details cannot be seen under a microscope, even at high resolution. The use of dyes is required through staining techniques, which provide contrast for observation.
Characteristics of a good quality bacterial smear
excellent contrast
PTo achieve excellent contrast there are sophisticated microscopes called phase contrast microscope, differential interference and dark field microscope. This type of microscope is used to observe bacterial structures such as sheaths and filaments, among others.
Staining is a simple technique to increase contrast that is achieved with a brightfield microscope. In this technique, different dyes can be used that significantly improve the observation under a microscope. The stains are carried out directly on the smears or extensions of the suspensions of microorganisms on the slides, previously dried and fixed.
good fixed
The Fixation is a technique used to preserve cell structures. It causes inactivation of microorganisms and adhesion to the glass of the slide. There are different fixing treatments: heat fixing and chemical fixing. Heat fixation: it is the most widely used method in the observation of bacterial smears. The technique consists of passing the bacterial suspension of the smear through the flame of a burner. This technique is capable of preserving the external morphology of the bacteria, but destroys their internal structures. Chemical fixation: chemical fixation uses chemical substances in preservation, such as formaldehyde or formol, ethanol and acetic acid, among others. The advantage of using chemical fixing agents is that the preservation of the internal cellular structures of the microorganisms is achieved.
good staining
LThe most common procedures for staining a previously dried and fixed smear are positive or simple staining, differential staining, and negative staining. There are also special techniques for staining particular cell structures (capsule, spores, flagella).
Positive staining or simple staining: hePositive or simple staining is the most widely used smear staining technique. It uses dyes that have the ability to bind to certain microbial structures, allowing them to be observed under a microscope. These dyes have chromophore groups (colored portion) in their chemical structure, with alternating double bonds and single bonds (conjugation). These links can in turn establish ionic or covalent bonds with some cellular structures. The dyes used in positive or simple staining are mostly chemical derivatives of aniline. (colored organic salts). On the other hand, among the dyes we can find some with basic pH and others with acid pH.
Basic dyes: eIn basic dyes, the chromophore group has a positive electrical charge. The vast majority of prokaryotic microorganisms have a neutral internal pH, and their cell surface is negatively charged. Through this electrostatic interaction, the chromophore binds to the cell and stains it. Examples of basic dyes are methylene blue, crystal violet, malachite green, basic fuchsin, safranin, among others.
Acid dyes: eIn acid dyes, the chromophore group has a negative electrical charge. These are used for staining proteins with positively charged amino groups. Examples of acid dyes are acid fuchsin, rose Bengal, Congo red, and eosin.
Differential staining: lThe differential staining technique consists of applying two dyes of different color or intensity, to distinguish different microorganisms under the microscope. Gram stain and acid-alcohol fast stain are the most widely used differential stains in bacteriology. Gram stain is used as a preliminary test to know the shape, size, cell grouping, as well as the type of cell wall. Using the Gram stain test, bacteria with a cell wall are classified into gram-positive bacteria and gram-negative bacteria.
Negative staining: en this technique, chemical dyes are used that do not penetrate the cell interior, but make the medium in which the microorganisms are found appear as a black background. In the negative staining technique, the smear is made with a drop of India ink or nigrosin suspension, which, after allowing to dry at room temperature, forms an opaque film to the passage of light. In this way, microorganisms are seen as bright shapes on a dark background.
Preparation
A. Smears
Wash the slides very well, dry with absorbent paper and label them. The label must indicate the content of the preparation, the date and the name of the person who processed it.
Light the burner and sterilize the inoculation loop in a flame until red hot.
Let the handle cool.
Take the tube of the bacterial culture, remove the stopper and quickly pass the mouth of the tube near the flame of the burner (flame).
Introduce the inoculation loop into the tube containing the bacterial culture and take the sample.
If the culture is in liquid medium, place the sample taken with the loop in the center of the slide and carefully spread it out in a circle approximately 2 cm in diameter.
Re-sterilize the inoculation loop.
Allow the smear to air dry.
Repeat steps 3-8 three times.
If the culture is in a solid medium, a drop of distilled water must be placed on the slide beforehand. This is done to mix a small sample of the culture taken with the inoculation loop, as directed in steps 2 to 5 (aseptic conditions).
Spread the diluted sample with the drop of water on the slide and repeat three times.
B. Fixation
Add two drops of methanol or absolute ethanol to the dried smears —from cultures in liquid medium.
Allow to air dry away from the lighter.
If the smear comes from a culture in a solid medium, the dry smear is fixed with heat, passing it 2 to 3 times quickly through the hottest area of the burner flame.
Touch the bottom of the smear with the back of your left hand (for right-handers. Otherwise, use your right hand) and check that it is cold.
C. Plain stain
Add 2 drops of the selected dye to the smear and leave it to act for the time required in the specific protocols for each dye (generally between 1 and 5 minutes).
Some dyes require the use of heat for activation, in which case one must be very careful when heating the slide in the burner flame (handle with tongs and avoid boiling). Overheating the smear can destroy the cells to be observed.
Remove excess stain by washing with distilled water from a small basin. Remove the wash water by gently tapping the slide on its edge, tilted on the work table.
Allow to air dry.
Depending on the type of observation, a coverslip is used or not at this stage. The coverslip protects and preserves the smear. If an oil immersion observation is made at this stage, coverslips are not used, but the smear cannot be preserved.
D. Definitive preservation of the smear
Immerse the smear successively in each of the solutions indicated below, for a minimum of 5 minutes. The purpose of these “baths” is to leave the smear completely dehydrated. Each reagent should be drained well before introducing the smear in the next bath.
The order of the dehydrating baths is as follows:
Ethanol 70%.
Ethanol 95%.
Pure acetone.
Mix acetone-xylol 1:1.
Xylol.
Then allow to air dry.
Mount the coverslip, preferably 22×22 mm, using Canada Balsam or other mounting medium.
References
Cappucino, JG and Welch, CT Microbiology: A Laboratory Manual. pearson.
Holt, JG The shorter Bergey’s Manual of Determinative Bacteriology. Baltimore: The Williams and Wilkins Co.