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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

what is fermentation example ?
Sonal Reply
is proceess in which an agent couses of an oganic substances breakdown into simpler substance,especially in aneorobic breakdown of suger into alcohol.
Okashat
is it better to study microbiology and then medicine it makes no difference to go directly to medicine?
Jessee Reply
Dray's mathdme cell wall konse color k hote he
Jinal Reply
what is dray's mathdme cell wall
Prabhat
I confused. please help me
Karen
just confused
Raj
that is dyar's method of cell wall staining
ART___
Cetylpyridinium chloride ionize in water to form positively charged cetylpyridinium and negatively charged  chloride ions. As bacterial cell wall is negatively charged, the positively charged ions of cetylpyridinium  are adsorbed on the cell wall making it positively charged. Subsequent treatment w
ART___
l don't understand it please explain it for me.
Karen Reply
epitopes are present on the surface of
Rohit Reply
at the tip of variable region on the antibody...where antigen and antibody binding sites combine...
Fiza
The term that is used refer to moving microbes under a microscope are referred to as?
Lee Reply
motile
ART___
Members of the genus Neisseria cause which of the folowing human diseases?
Farah Reply
genital infections
Kamaluddeen
gonorrhoea
sandip
gonorrhoea
Jessee
4. Which of the following specimens should not be refrigerated? a. Urine b. Urogenital swab
Zahraa Reply
urine
Muuse
urine
Agatha
Urine
Tean
urine
Yasser
Urine
Ebtehal
urine
anamika
urine
Puja
urine
Inemesit
urine
Samuel
Urine
Muhammad
Details about McConkey agar
Muhammad
urine
SK
urine
urine can be refrigerated for 24hours but only in sealed container...or else the microbes will multiply itself
ART___
what is bacteria
anamika Reply
a member of large number of unicellular microorganism which have cell wall but lack of cell organelles an oranised nucleus including somewhat can cause disease
Sukhdeep
Bacteria are usually composed of one cell onl to that are neither plants nor animals, microscopic, that may cause diseases or may be beneficial(in gut)... it depends upon their weapons. Nearly all animal life is dependent on Bacteria for their survival
Fiza
thanks
anamika
what factor make bacteria colony large and how could we sterlise it in large scale
fatty
nutrient concentration temp gaseous conc ph ion or salt concentration mositure condition factors contribute to make large colony. by autoclaving we will sterilize bactetia
Sukhdeep
Colony is actually visible growth of Bacteria that is as a result of suitable environment for growth i.e optimal conditions for growth, temperature, moisture etc. there're many methods to get rid of bacteria. If We stop giving them optimal conditions for living Bacteria will die soon .
Fiza
what's the difference between an antigen and a pathogen?
Pathogens are organisms that cause disease in other organisms whereas Antigen is a part of a pathogen that triggers the immune response..
Rajat
so it is the antigen that dendritic cells present to the T cells and not the pathogen itself?
no no antigen are the west product or part of the pathogen. in such case bacteria it self fight with over immune response & in another case bacteria release antigens
vasava
& other antigen like pollan grain, dust particles etc.....
vasava
pathogen are microbes that can infect the body and causw illness....antigens are the part of pathogens that alert the body to an infection
Sukhdeep
antigen is a part of blood and pathogen is foreign particle which causes diseases
Yogyata
antigen could be non microorganism.... where as pathogen is mixroorganism
tadesse
Thanks
Karen
a pathogen is a disease causing organism while an antigen is a protein in the white blood cells which combats pathogens.
Jessee
pathogens produce antigen which attacks the cell of the host ...the antigens are proteins and are present on the surface of the pathogen
ART___
a pathogen produce antigen which attacks the cell of the host while antigens are proteins in the white blood cells which combat pathogens
Madrine
what type of widal test
sobhit Reply
this test determine for typhoid in this test if H,O antigen are present that indicate the positive test bac. are salmonella typhy
vasava
what h.o denotes
Iqra
o: body of bacteria, h: flagellate
Explain Mould
Chinenye Reply
Explain mycoses and it's classification
Chinenye
why do we have hiccups?
Manisha Reply
shakey diaphragm
Curlisse
The antibody binding site is formed primarily by:
Asalla Reply
How many types of MICROORGANISMS do we have?
Hope Reply
Hello friends
Hope
microorganisms are divided into seven type Bacteria archaea protozoa algae fungi virus and multicellular animal parasites
Raj

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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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