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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 pathogenic
kelvin Reply
biological pathogen is anything that can produce disease. a pathogen may also be referred to as an infectious agent, or simple a germ.
Dejene
Thanks
kelvin
explain 3 points why gender is relevant to health.
kelvin
wat causes sor throat
Ezeama Reply
Streptococcus
Rajat
Streptococcus
Panda
Gram positive
shamim
streptococcus pyrogenes
Sherma
group A streptococcus pyogenes. But not the only one.
Carlos
does the bac of s.aures gives choronic diseas in bone?
Elyas
causes virus bac group A strep allergies smoking
Elyas
truth also virus
Shania
what z microbiology
Mwelwa Reply
microbiology is all about the scientific study of microorganisms, their life changes, their life span, structural punctuality and their phenomenon.
Ridwan
thanks
micah
Best scope in microbiology
Rahul
wat abut skin
Elyas
whatis scope
Amare
what happened when someone died and what happened to the blood
Franca Reply
nice question
Attari
when you die you have no memory and the blood dry off
Falere
what microorganisms is all about?
Ridwan
And how many does the microorganisms is classified?
Ridwan
How are microbs organized
Saskia
what is endoparasites and exo parasite and their example
OGEDE Reply
Endo : inside the body Exo/ecto : outside or on the body
Paul
examples please
OGEDE
Louse for exo/ecto Helminths (worms) for endo
Paul
what's prokaryotic
mohaiminul Reply
A prokaryotic cell is a cell with no membrane bounds organelles
Paul
what are the main effect of parasite?
yunusa Reply
nutritional group of micro organism
Mana Reply
what are the charateristic bacteria, eukaryotes, and viruses have in commom
Ami Reply
what type of bacteria ismor serious
Amare
D N A ar purno rup ki
Ruba Reply
বাংলায় দেখবো DNA এর পূর্রন্যরুপ কি
Ruba
what's microbiology
micah
micro biology is the study of micro organisms,those being unicellular multicellular or a cellular
aniisha
deoxyribonucleic acid is a long molecule that contains our unique genetic code.
aniisha
Does the parasite/parasitology are under microbiology or not
yunusa
Define bacteria
Kainat
Bacteria are a type of biological cell & they constitute a large domain of prokaryotic microorganisms.
raisa
what kind of microbiology
Mira
hello what is main function of T and B cells
Ishaq Reply
T cells are involved in cell-mediated immunity while B cells are primarily responsible for humoral immunity (relating to antibodies)
Janet
what is hepatitis B
Sunday
it's viral infection that attacks the liver and can cause both acute and chronic diseases. the virus is most commonly transmitted from mother to child during birth and delivery, as well as through contact with blood or other body fluids...
Dejene
What are fundamental experiment of microbiology?
Lucky Reply
study of microscopic organisms, unicellular and multicellular
Falere
what are the characteristics of bacteria, viruses, and eukaryotes
Ami
bacteria-are living organism. contain a peptidoglycan/lipopolysaccharide cell wall. VIRUS-are considered as organic structure which interact with living organism don't have acell wall. they have many differents like bacteria size -large(1000nm) virus size-smaller(20-400nm). bacteria ribosomes presen
Dejene
virus ribosomes absence. bacteria living organism. virus b/n living and non-living things
Dejene
eukaryotic cell has a true membrane bond nucleus and has other membranous organelles that allow for compartmenalization of function
Dejene
This answer is wrong
Jeffrey Reply
who discovered the growth curve
Joy Reply
Example of coccus bacteria
Nweke Reply
staphylococcus aureus
Elton
MRSA any bew information
Satish
pl. read any new information about MRSA
Satish
Any stru tural finding about novel corona virus
Satish

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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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