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Learning objectives

  • Differentiate between simple and differential stains
  • Describe the unique features of commonly used stains
  • Explain the procedures and name clinical applications for Gram, endospore, acid-fast, negative capsule, and flagella staining

In their natural state, most of the cells and microorganisms that we observe under the microscope lack color and contrast. This makes it difficult, if not impossible, to detect important cellular structures and their distinguishing characteristics without artificially treating specimens. We have already alluded to certain techniques involving stains and fluorescent dyes, and in this section we will discuss specific techniques for sample preparation in greater detail. Indeed, numerous methods have been developed to identify specific microbes, cellular structures, DNA sequences, or indicators of infection in tissue samples, under the microscope. Here, we will focus on the most clinically relevant techniques.

Preparing specimens for light microscopy

In clinical settings, light microscopes are the most commonly used microscopes. There are two basic types of preparation used to view specimens with a light microscope: wet mounts and fixed specimens.

The simplest type of preparation is the wet mount , in which the specimen is placed on the slide in a drop of liquid. Some specimens, such as a drop of urine, are already in a liquid form and can be deposited on the slide using a dropper. Solid specimens, such as a skin scraping, can be placed on the slide before adding a drop of liquid to prepare the wet mount. Sometimes the liquid used is simply water, but often stains are added to enhance contrast. Once the liquid has been added to the slide, a coverslip is placed on top and the specimen is ready for examination under the microscope.

The second method of preparing specimens for light microscopy is fixation . The “fixing” of a sample refers to the process of attaching cells to a slide. Fixation is often achieved either by heating ( heat fixing ) or chemically treating the specimen. In addition to attaching the specimen to the slide, fixation also kills microorganisms in the specimen, stopping their movement and metabolism while preserving the integrity of their cellular components for observation.

To heat-fix a sample, a thin layer of the specimen is spread on the slide (called a smear ), and the slide is then briefly heated over a heat source ( [link] ). Chemical fixatives are often preferable to heat for tissue specimens. Chemical agents such as acetic acid, ethanol, methanol, formaldehyde (formalin), and glutaraldehyde can denature proteins, stop biochemical reactions, and stabilize cell structures in tissue samples ( [link] ).

Photograph a shows a slide sitting on a flat heating surface. Photograph b shows a person holding a slide against a heated metal cylinder. Photograph c shows a bit of tissue in a container of clear liquid. Caption reads Fixing tissue: fixation to preserve tissue and maintain life-like structure; place into fixative (eg 10% formalin).
(a) A specimen can be heat-fixed by using a slide warmer like this one. (b) Another method for heat-fixing a specimen is to hold a slide with a smear over a microincinerator. (c) This tissue sample is being fixed in a solution of formalin (also known as formaldehyde). Chemical fixation kills microorganisms in the specimen, stopping degradation of the tissues and preserving their structure so that they can be examined later under the microscope. (credit a: modification of work by Nina Parker; credit b: modification of work by Nina Parker; credit c: modification of work by “University of Bristol”/YouTube)

Questions & Answers

it could be hormonal in balanced kindly see a doctor thanks
Precious Reply
U mean u have never had Ur period before?
Tee Reply
pregnancy
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how many months does you notice that problem
pavan Reply
I am 21 years old and I haven't seen my period,what could be the cause?
Faith Reply
Please what is the solution to the problem?
Faith
abnormal cytobacteria ukrine
dzaram Reply
my period dos'nt flow as normal Pls what could be the cause?
Chinelo Reply
No some are beneficial & others are pathogenic
Diuniceous Reply
are all bacteria dangerous
Lorretha Reply
What is a bacteria?
Nang Reply
PLEASE WHAT IS CULTURE ?
Emmanuella Reply
Which type of cytoskeletal fiber is important in the formation of the nuclear lamina?
shaletta Reply
Nice, explanation about Gram's bacterial differential procedure
surendra Reply
Beer, cheese, bread, are orange juice is not fermentation
shaletta Reply
describe gram staining
Aditi Reply
if I'm not mistaken(no googling) it's the testing of bacteria to see whether it is gram positive or negative... it is clinically useful because if you have a gram positive person you don't want to give them gram-negative medicine
Coach
it is a procedure in which we have know about bactrria either gram positive or gram negative after cultivation of bacteria on agat plates we put crystal voilet then iodine then de colorizer and then if not retain voilet colur so put safranin for gram negative bacterai in case of retaining voilet
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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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