Bacterial Smears Are Fixed before Staining to?
It is important to heat fix the bacterial smear before staining so as to, kill the bacteria, firmly adhere the smear on to the microscopic slide to prevent washing off during staining, and to allow the sample to readily take up the stain. Reference:
What is the purpose of heat- fixing the smear?
It helps the cells adhere to the slide so that they can be stained. The purpose of heat fixing is to kill the organisms without serious distortion. They adhere better to the slide and also take up dye more easily. Fixation process
Fixation is usually the first stage in a multistep process to prepare a sample of biological material for microscopy or other analysis. Therefore, the choice of fixative and fixation protocol may depend on the additional processing steps and final analyses that are planned. For example, immunohistochemistry uses antibodies that bind to a specific protein target. Prolonged fixation can chemically mask these targets and prevent antibody binding. In these cases, a 'quick fix' method using cold formalinfor around 24 hours is typically used.
Types of fixation
There are generally three types of fixation process:
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. Heat fixation generally preserves overall morphology but not internal structures. Heat denatures the proteolytic enzyme and prevent autolysis. Heat fixation cannot be used in the capsular stain method as heat fixation will shrink or destroy the capsule (glycocalyx) and cannot be seen in stains. Perfusion: Fixation via blood flow. The fixative is injected into the heart with the injection volume matching cardiac output. The fixative spreads through the entire body, and the tissue doesn't die until it is fixed. This has the advantage of preserving perfect morphology, but the disadvantages that the subject dies and the cost is high (because of the volume of fixative needed for larger organisms) Immersion: The sample of tissue is immersed in fixative of volume at a minimum of 20 times greater than the volume of the tissue to be fixed. The fixative must diffuse through the tissue to fix, so tissue size and density, as well as type of fixative must be considered. Using a larger sample means it takes longer for the fixative to reach the deeper tissue. Best in a slight vacuum. Chemical Fixation
In this process, structures are preserved in a state (both chemically and structurally) as close to living tissue as possible. This requires a chemical fixative that canstabilise the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble. Types of Chemical Fixatives
Crosslinking fixatives - Aldehydes
Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. By far the most commonly used fixative in histology is formaldehyde. It is usually used as a 10% Neutral Buffered Formalin (NBF), that is approx. 3.7%-4.0% formaldehyde in phosphate buffered saline. Because formaldehyde is a gas at room temperature, formalin-formaldehyde gas dissolved in water (~37% w/v)-is used when making the former fixative. Paraformaldehyde is a polymerised form of formaldehyde, usually obtained as a fine white powder, which depolymerises back to formalin when heated. Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine. Its effects are reversible by excess water and it avoids formalin pigmentation. Other benefits include: Long term storage and good tissue penetration. It is particularly good for immunohistochemistry techniques. Also the formaldehyde vapour can be used as a fixative for cell...
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