<< Chapter < Page Chapter >> Page >

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

a solvent isopropyl myristate is use for the pre-treatment of?
Nasir Reply
what is microbiology
Ayeniyi Reply
microbiology is the study of all living organisms that are too small to be visible to the naked eyes, examples bacteria and fungi etc
Iheme
study of microorganism such as becteria etc
Muhammad
study of microorganisms and their effect on organisms life
Aqsa
characteristic of Gram negative bacteria
jane Reply
Characteristics of Gram Negative Bacteria As with Gram positive bacteria, Gram negative bacteria also contain the peptidoglycan polymer in their cell wall. While this polymer is thin (2 to 4 nanometers in thickness with just about 3 layers of peptidoglycan) in Gram negative bacteria, it's also com
Kaviya
it's also composed of long glycan strands that are cross-linked by peptide molecules. This composition serves a number of functions including protecting the bacterial cell from lysis
Kaviya
Good shot
Enoch
Thanks 😊
Kaviya
what was Hans Christian Gram's supported in the modern Microbiology?
Wilson Reply
what is microbial growth
Chisa Reply
The organism responsible for vulva ulcers
nyiter Reply
Why are vascular pathogen poorly communicable from person to person?
Aj Reply
Most vascular pathogens are poorly communicable from person to person because they need a medium to be communicated i,e a vector that would carry them from one person to other
Kaviya
what's the habit of protista
Afieahngwi Reply
They show both autotrophic and heterotrophic mechanisms...
Swetha
thanks
Afieahngwi
welcome...
Swetha
let me mention some water. Air .Food and so on
Gattiek Reply
causes of infectious diseases
Afieahngwi Reply
water.Air
Gattiek
infectious disease are caused by pathogenic micro organisms like bacteria ,fungi..
Swetha
What is pasteurization?
Wilson
are fungi prokaryote or eukaryotes?
Afieahngwi Reply
fungi are eukaryotes.
Swetha
All fungi are eukaryotes. Even micro fungi.
Lad
have..complex cellular organization and membrane bound nucleus ...and..also... having loops of DNA( like plasmids) as.bacteria
Swetha
what enzyme replaces rna nucleotides with dna nucleotides during replication?
Remi Reply
an enzyme called DNA ligase.
Jael
describe the acid fast staining procedure used in the diagnosis of tuberculosis
Salma Reply
bacterial morphology
lf_ Reply
what is the difference between biogenesis & abiogenesis
Mayuri Reply
biogenesis is when living comes out from other living things as a result of reproduction while a biogenesis is the process where living things comes out from non living things
Usman
living things come form other form living things is biogenesis. ....right?
Mayuri

Get the best Microbiology course in your pocket!





Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
Google Play and the Google Play logo are trademarks of Google Inc.

Notification Switch

Would you like to follow the 'Microbiology' conversation and receive update notifications?

Ask