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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 rickettsia
DENNIS Reply
what is microbiology
Erasto
state the theory of spontaneous generation of micro oranisms and germ theory of disease
UKAMAKA Reply
what are the advantages of high note numerical aperture
Genius Reply
list if non flagellated pritozoa
Mepung Reply
Can someone that's understanding of the Kreb Cycle please explain & breakdown it down to me in the simplest way without giving me the dictionary version or Google version. Basically in there own words of knowledge....!
Kisha
ok
lucas
please can someone help explain positive and negative feedback in simple term
Gum
negative feedback is the arresting of reaction or reverse of the reaction according to the response and postive feed back is the direct response without reversing or arresting a reaction.
Greet
OK
umar
pls can someone explained Kinney stone in memorising in shot time
umar
what tyoebif microorganism will be killed by antibiotic trwatmeant
Mary Reply
I don't really don't understand aerobic respiration anaerobic respiration and fermentation. Can someone explain & breakdown this in the simplest way please.
Kisha
Anaerobic Respiration in which foodstuffs are partially oxidized, with the release of chemical energy, in a process not involving atmospheric oxygen, such as alcoholic fermentation, in which one of the end products is ethanol.
Muhammad
aerobic respiration A type of respiration in which foodstuffs are completely oxidized to carbon dioxide and water, with the release of chemical energy, in a process requiring atmospheric oxygen.
Muhammad
please can someone explain what pseudomonas species and biofilm is?
uju
Fermentation is the growth of cells or microorganisms in bioreactors (fermenters) to synthesize special products. Fermentation in biochemistry refers to the biodegradation of carbon compounds by cells or organisms under anaerobic (lack of oxygen) conditions.
Muhammad
Please under which conditions does pathogens become established in the human tissues and can cause diseases
Atambilla
please the life cycle of plasmodium parasite
mavis
who is John Needham
Mary Reply
John Needham is one of the researcher in microbiology. He also experimented when scientists did not believe animals could arise spontaneously ,but did believe microbes could.
Gum
okay is he late
Mary
by the way what are the list of courses offered by a newly admitted student for microbiology
Mary
Needham's experiments with beef gravy and infusions of plants material reinforced this idea.
Gum
what drugs are given to a person with Otis nerve problem(ear problem)
Gum Reply
good morning to you all.
Muhammad Reply
Dr.A.K.S.P.G. college Akbarpur Ambedkar Nagar
Rajan
Shailesh Vishwakarma BSc 1st year
Rajan
differentiate between eukaryotes en prikaryotes
AKANKWATSA Reply
eukaryotes are organisms with complex cellular structure(presence of nucleus and mitochondrial) like plants and animals whilst prokaryotes are organisms with simple cellular structure (absence of nucleus, mitochondrial) like arthropods.
Seidu
hlo
Shagufta
hii
dr
hello sir
pankaj
I am Pankaj from bihar
pankaj
I am study in NDRI karnal in btech in dairy
pankaj
I am Pankaj from bihar
pankaj
thanks am studying biomedical sciences in zambia.chikankata
Sharon
Am a studying Disease Control in Ghana(kintampo)
Seidu
I'm studying Environmental health in Zimbabwe 🇿🇼
trust
am Joyce mwale studying nursing in Lusaka Zambia
Joyce
hi pankaj
Shailesh
hi frds iam from India
Nevil
IAM studying degree (micro biology)
Nevil
Hi I am microbiologist
Muhammad
Hi friends I'm from Nigeria studying Microbiology
uju
i am biotechnologist from pakistan
Saddam
hi feds iam from india
Shibanjan
hi
Krishna
india
Krishna
hy frnds am from Uganda
AKANKWATSA
am studying nursing/midwifery n kampala
AKANKWATSA
I'm Gum Joseph Kakuu from Ghana,I'm studying RGN in nursing Jirapa.
Gum
I'm Hellen from Kenya studying applied biology
Hellen
I live in india
Krishna
am studying biotechnology
Krishna
am maryjoy uwaoma from Nigeria,I am studying microbiology in NSUKKA NIGERIA
Mary
Nice meeting You people hear
Seidu
thanks you.
Gum
can someone in there own words in the simplest way breakdown what aerobic respiration anaerobic respiration and fermentation means please and not from the dictionary version...!
Kisha
what are the cultivation and identification of microorganisms
Algel Reply
ransport media.basic culture media.enriched media..differential media .indetification media
AKANKWATSA
algel th above are some v th basic medias for bacterial growth
AKANKWATSA
factors that can Prone the host to get disease
Linda Reply
what are the factors that prones host to get disease
Linda
HELLO
Hashim
Not sure about it, can you tell us Linda?
KINGSFORD
please I want to know the factors that prones host to get disease
Linda
please Linda we have internal and external factors. internal factors are within our body cavities.eg mother having TB giving it to her child,and the others. external factors are within our surrounding environment which include our daily activities(bathing, buying food and the others)
Gum
I don't understand aerobic respiration anaerobic respiration and fermentation. Can someone that do give me a short & simple breakdown.
Kisha
pliz kisha ..aerobic respiration is th typ v repiration whre organisms use oxgyen for survival while anaerobic respiration is whre organisms survive without oxgyen at all.these use carbondioxide
AKANKWATSA
Guys pls answer me this question fast... What are the purpose of infection prevention in 50 words..
RAPHERA Reply
The purpose of infection and prevention is a scientific approach and practical solution designed to prevent harm cause by infection to Pt and health worker
what is the sequence of kingdom concept?
isha Reply
what is immunity
Algel
what are the classification and identification of microorganisms
Algel
whats anatomy
abdiqani
and physiology
abdiqani
anatomy is the study of the human structure
physiolog is the study of the human body function
Anatomy and physiology is the study of the human body parts and their functions.
Kwame
who's the father of Microbiology
The definition you gave is not for the two
Samuel
leeuwenhoek is the real father of microbiology
abdiqani
anatomy is the study of the structure and relationship between body part while Physiolog is the study of the function of body part
thats real definition
abdiqani
I am sure about Louis pasteur been the real father of microbiology
Mary
microbiology father is marcelo
DENNIS
sorry the father is Anthony Van Leeuwenhoek
DENNIS
The father is Marcello Malpighi
DENNIS
Anatomy and physiology is the study of the body structures and how they relate to each other
Unbeatable
which drawing in figure 4.1 is a tetrad
adnaloy Reply
OK I hear
stella
syphilis a reproductive disease?
Ulamila Reply
yaa but not exactly
Medhini
STD
Medhini
what are the possible disease cause by virse?
Victor
what is improtance thing we need to know about fungi!
Victor
toxins,ancient origin ,and diseases by fungi are very hard to cure
kalyani
STD
Uko
The disease cause by virus is HIV/AIDS
thanks
Victor

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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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