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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

write the life cycle of HIV
Firomsa Reply
describe the internal and external structure of prokaryotic cell in terms of there appearance and functions
Lenia Reply
compare and contrast similar structures found in prokaryotic and eukaryotic cells
Lenia
control of microorganisms
ISTEFAZUL Reply
how can a doctor treat a person affected by endospore forming bacteria in his/her wound?
Mambo Reply
a 28 years old woman come to your clinic with complain of fever painful genital blisters which express clear fluid when ruptured burning sensation around the bristers what is the diagnosis?
Ramadhani Reply
genital herpes caused by a virus called herpe simplex virus
Doris
what is the reference between selective medium and differential medium?
Tony Reply
the micro flora of air is transient why
Princess
Hello.... Am new here
essien Reply
welcome essien
Muhammad
welcome essien
Beka
classification of gram positive
lissa Reply
classify gram positive
lissa
catalase test is done to differentiate between staph and strep. as staph is catalase positive while strep is catalase negative then staph is differentiate further on coagulase positive and coagulase negative. staph aureus is coagulase positive while staph epidermidis is coagulase negative.
Muhammad
gram positive staph is further differentiated on sensitivity test and manitol salt fermentation test while gram positive strep is differentiated on hemolysis pattern
Muhammad
more about gramm positive
lissa
please
lissa
ok
Blessing
am back any gist
Blessing
taxonomy
Rahul
can i use the graph of bacterial growth for my master's thesis?
Christoph Reply
facultative anaerobic bavteria gives uniform turdibity in nutrient broth why?
Princess Reply
oils and waxes are not sterilized in autoclave
Princess
what is the function of paraffin
Muhammad
@ MAHI because they can grow all over the media from surface to the bottom as they can utilize oxygen or conduct fermentation in its absence.
Online
2.@MAHI Autoclave uses steam under pressure. The steam cannot penetrate through oil and wax. Thus, dry heat sterilization is preferred than autoclave .
Online
thnku
Nadiya
how nutirent agar can converted into blood agar
Princess
thank you
Princess
take a nutrient agar ........and add 5ml blood and put in it ...........!
Nadiya
nutrient agar +blood 5ml+ distilled water =blood agar .
Nadiya
mixed wellllll
Nadiya
hi
Rahul
why agar is not a neutrient source?
Shimul
because it's a general purpose agar only use for growing bacteria
Richa
way is antigen
opaleye
what is antigen
opaleye
antigen is a foreign body that cause activation of antibody?
ASNAKE
Antigen is a toxin or other foreign substance which induces an immune response in the body, especially the production of antibodies.
Shimul
thanks for that
opaleye
what is pharmacology
Luyenu
a science that studies about the drug
ASNAKE
hello ever one
Sayid
y
Abdul
Work hard
Anigor
y
Abdul
thanks alot
Luyenu
What is the major different between gram positive and gram negative bacteria.
Asikur
bacteria kya hai
Mala
content of their cell wall
Doris
koi mujhse basic microbiology ki study k Lea book suggest kro .
Richa
Harley prescott
Vikas
what are the five predisposing factors of opportunistic infections
Asher Reply
1. long term exposure to diseases, that the body immunity weakens.
prosper
2. Nature Of some diseases to weaken body immunity such as, AIDS.
prosper
3. Use of immuno-suppresive drugs.
prosper
anyone... need a hand here 🙋😞
prosper
Low immune is the major cause eg for kaposis sarcoma
Davismith
suitable conditions e.g temp,
Muhammad
what os gnormal flora
kifayat Reply
morphological and genetic classification of bacteria
Potter Reply
what is the morphogical
Bupe
h
Sudhir
morphologycal is defined as relating to the branch of biology that deals with the form of living organisms,and with relationship between their structures.
Nadiya
relating to the form or structure of things .
Nadiya
what is the meaning of lessions
oluwa
i am frim Afghanistan.
wahidullah Reply
iam from iraq
Haidar
and i'm from pakistan
Muhammad
Nice
Amina
what's about the qualification
Muhammad
Hlo
Asiya
Hmm
Number
Almustafa Muhammad from Nigeria
Mujtafa
am ustaz Abdulsalam from Ghana
ustaz
am Bupe Chifita from Zambia
Bupe
what is microbiology
Bupe
is the study of invisible micro organism
Mujtafa
microbiology is the study of very too small organisms which we can't see with naked eyes ......in other words ....study of microorganism is called microbiology.
Nadiya
Hello! How Can Microorganisms Be Isolated From The Skin?.. 5 ways if possible. (briefly)
prosper
from skin swabbing is the prefer method worldwide
Muhammad
thanks Muhammad Nauman very much!
prosper
hi
Meek
hi Meek
prosper
are there any other ways pals?
prosper
hi prosper
Meek
hello Meek Mild.
prosper
Any question or ideas, Meek?😊
prosper
and if a patient have any blister or abscess on the skin then first cut the blister or abscess. inside material is taking by syringe.
Muhammad
in case of fingernail or toenails simply cut a small piece of nail and culture on the appropriate media at a required temperature and time
Muhammad
Again, thanks Mr. Muhammad! I appreciate the help.
prosper
you welcom prosper 😊
Muhammad
what are the media use for skin swab?
Sieh-Mlanwin
blood and macckonkey mostly usededia for skin bacteria
Muhammad
used media*
Muhammad
@ SIEH mostly fungal infection occurs in skin so SDA ( Sabouraud Dextrose Agar) or MEA ( Malt extract agar) are common.
Online
thanks
Sieh-Mlanwin
Microbiologicam source of vitamins
Bachi Reply
what do you mean?
Md
that's example?
Md
hi everyone
Muhammad
such as. c/s media
Md
hmm hlw
Md
just tell me about that ?
Md
where are u from
Muhammad

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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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