Microbiology and Parasitology Vincentian Learning Module

Basic Laboratory equipment and procedures

LESSON 10 (Staining procedure and culture media)

OVERVIEW
Knowing the proper procedure and tools opens any laboratory procedure
to a successful experiment.
In this module you are task to familiarize yourself with the different
staining techniques in media culture.

LEARNING OUTCOMES

At the end of this module, you will:

1. Determine laboratory procedures;
2. Familiarize the staining process; and
3. Showcase your own understanding of the staining process and its
utilization.

LEARNING CONTENT AND SELF ASSESSMENT QUESTIONS (SAQs)

Because individual microbes are typically too small to be seen with the
naked eye, microbiology relies on technology to augment our natural
senses of perception. Pasteur and Koch, for example, had fewer tools at
their disposal than modern microbiologists, making their discoveries and
innovations all the more impressive. Many applications of technology will
be explored in depth in later chapters of this text, but for now, here is a
quick overview of some of the most basic microbiology lab tools.

 Microorganisms in a lab setting are grown on growth media. Some

of the media are liquids, while others are solids or gels.
Microorganisms can grow and reproduce in a growth medium that
contains nutrients such as water, various salts, a source of carbon
(such as glucose), and a source of nitrogen and amino acids (such

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as yeast extract). Ingredients in a growth medium can be tweaked

to grow specific types of microbes or to help distinguish or identify
multiple microbes growing in close proximity.
 Test tubes are rounded-bottomed, open-top cylindrical plastic or
glass tubes. Microbes can be grown in broth, semisolid or solid
growth media with them.

 A Petri dish is a flat-lidded dish that is typically 10–11 centimeters

(cm) in diameter and 1–1.5 cm high. Petri dishes made out of
either plastic or glass are used to hold thin layers of solid media,
usually solidified with agar.
 A Bunsen burner is a metal apparatus that creates a flame that
can be used to sterilize pieces of equipment. A rubber tube carries
gas (fuel) to the burner. In many labs, Bunsen burners are being
phased out in favor of infrared microincinerators, which serve a
similar purpose without the safety risks of an open flame.
 An inoculation loop is a handheld tool that ends in a small wire
loop The loop can be used to streak microorganisms on agar in a
Petri dish or to transfer them from one test tube to another. Before
each use, the inoculation loop must be sterilized so cultures do not
become contaminated, and re-sterilized after the transfer.
 An inoculation needle is another handheld tool that is used for
specific solid or semi-solid inoculations. It looks similar to the loop
except that it is a straight point. It can help to determine whether
an organism is motile (moves). Like the inoculation needle, it needs
to be sterilized before every use, and afterwards too.
 Sterile cotton swabs are another common tool of microbiology,
useful for collecting samples from surfaces. However they are not
able to be reused.
 Microscopes produce magnified images of microorganisms, human
cells and tissues, and many other types of specimens too small to
be observed with the naked eye. Microscopes will be discussed
more in chapter 3.

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 Stains and dyes are used to add color to microbes so they can be
better observed under a microscope. Some dyes can be used on
living microbes, whereas others require that the specimens be fixed
with chemicals or heat before staining. Some stains only work on
certain types of microbes because of differences in their cellular
chemical composition. 

Inoculating and Incubating Media

Aseptic technique is always observed when transferring and inoculating

media after applying a label. The inoculation is the introduction of a
microbe into "clean" media (media free from other microbes). The
instrument used is always sterile or sterilized in a microincincerator or
Bunsen Burner first, then used to pick up the sample and applied to the
new media. The instrument should either be disposed of or re-sterilized
after each inoculation. This is the best procedure to try to ensure a pure
culture- a culture with only one microbe growing. Mixed cultures
(purposefully adding multiple organisms) are occasionally use to show
how isolation streaking can be used to separate organisms. The goal is to
avoid a contaminated culture, where unwanted microbes are growing
next to or on top of the desired organism.

After the inoculation, the microbe will need to be incubated at its

preferred temperature and with its preferred oxygen gas exposure for a
number of hours or days. Generally the incubation times are shorter for
prokaryotes than for eukaryotes. Test tubes are always incubated in a
rack or other holder upright, while plates are incubated upside down.

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Describing the Growth of Microbes in Media

Once a microbe has been grown on a plate or in a test tube, they need to
be observed for various characteristics. Broth cultures are the easiest to
describe as they have the fewest options. Clear broth generally indicates
no growth. Cloudy (turbid) broth has growth in it, obscuring the light
coming through. Sometimes this growth is only at the top of the tube
(pellicle) or only at the bottom (sediment). The last possibility is that the
broth has flakes of growth throughout.

Figure 3. The caps are missing, but this image shows the bottom of several
possible growth patterns in broth. (credit: Microbiology Lab I, Libretext Ancillary
material)

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For growth on plates, the technique used to inoculate the plate does
make a difference. If the plate was inoculated using a single line
inoculation, generally the optical property (opaque, translucent or
transparent), overall texture (dry, moist or oily) and the colors of both the
microbe and the media are observed. For isolated colonies, especially
when identification is desired, more observations are required. When
observing isolated colonies we generally describe the following:

 general size (small, medium or large can be used or you can

measure its diameter in mm)
 general shape/form (circular, irregular, filamentous, rhizoid)
 optical properties (opaque, translucent or transparent)
 the color of the colony (or colors)
 any discoloration of the agar under or around the colony
 the edge or margin of the colony is described in terms of how
smooth it is.
 the surface should be described in terms of both texture (dry,
moist or oily) and smoothness of the surface (smooth, contoured,
radiating, concentric, rugose)
 elevation as seen from a side-on or profile (raised, convex, flat,
umbonate, crateriform)

Common terms used to describe isolated colonies. (credit form, elevation and
margin: McLaughlin and Peterson, Queensborough Community College; credit
surface of colony: Gray Kaiser, Community College of Baltimore County)

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Laboratory Biological Safety Levels

For researchers or laboratory personnel working with pathogens, the

risks associated with specific pathogens determine the levels of
cleanliness and control required. The Centers for Disease Control and
Prevention (CDC) and the National Institutes of Health (NIH) have
established four classification levels, called “biological safety levels”
(BSLs). Each BSL requires a different level of biocontainment to prevent
contamination and spread of infectious agents to laboratory personnel
and, ultimately, the community.

BSL-1 agents are those that generally do not cause infection in healthy
human adults. These include noninfectious bacteria, such as
nonpathogenic strains of Escherichia coli and Bacillus subtilis, and
viruses known to infect bacteria (phages) or animals other than humans,
such as baculoviruses (insect viruses). Because working with BSL-1
agents poses very little risk, few precautions are necessary. Laboratory
workers use standard aseptic technique and may work with these agents
at an open laboratory bench or table, wearing personal protective
equipment (PPE) such as a laboratory coat, goggles, and gloves, as
needed. Cleaning the area before and after working with a disinfectant
like bleach is generally required. Other than a sink for handwashing and
a door separating the laboratory from the rest of the building, no
additional modifications are needed.

BSL-2 through BSL-4 organism require higher amounts of containment.

These will be discussed further in chapter 11.

Handwashing the Right Way

Handwashing is critical for public health and should be emphasized in a
clinical setting. For the general public, the CDC recommends
handwashing before, during, and after food handling; before eating;
before and after interacting with someone who is ill; before and after
treating a wound; after using the toilet or changing diapers; after
coughing, sneezing, or blowing the nose; after handling garbage; and
after interacting with an animal, its feed, or its waste.
Figure 1.6.61.6.6 illustrates the five steps of proper handwashing
recommended by the CDC.

Handwashing is even more important for health-care workers, who

should wash their hands thoroughly between every patient contact, after
the removal of gloves, after contact with bodily fluids and potentially
infectious fomites, and before and after assisting a surgeon with invasive
procedures. Even with the use of proper surgical attire, including gloves,

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scrubbing for surgery is more involved than routine handwashing. The

goal of surgical scrubbing is to reduce the normal microbiota on the
skin’s surface to prevent the introduction of these microbes into a
patient’s surgical wounds.

There is no single widely accepted protocol for surgical scrubbing.

Protocols for length of time spent scrubbing may depend on the
antimicrobial used; health-care workers should always check the
manufacturer’s recommendations. According to the Association of
Surgical Technologists (AST), surgical scrubs may be performed with or
without the use of brushes 

(a) The CDC recommends five steps as part of typical handwashing for the
general public. (b) Surgical scrubbing is more extensive, requiring scrubbing
starting from the fingertips, extending to the hands and forearms, and then up
beyond the elbows, as shown here. (credit a: modification of work by World
Health Organization)

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END OF LESSON ASSESSMENT

Name _________________________________________________
Section _______________________________________________

PART 1. Case Scenario Assessment

A Year End Picnic

The microbiology department is celebrating the end of the school year in

May by holding its traditional picnic on the green. The speeches drag on
for a couple of hours, but finally all the faculty and students can dig into
the food: chicken salad, tomatoes, onions, salad, and custard pie. By
evening, the whole department, except for two vegetarian students who
did not eat the chicken salad, is stricken with nausea, vomiting,
retching, and abdominal cramping. Several individuals complain of
diarrhea. One patient shows signs of shock (low blood pressure). Blood
and stool samples are collected from patients, and an analysis of all
foods served at the meal is conducted.

Bacteria can cause gastroenteritis (inflammation of the stomach and

intestinal tract) either by colonizing and replicating in the host, which is
considered an infection, or by secreting toxins, which is considered
intoxication. Signs and symptoms of infections are typically delayed,
whereas intoxication manifests within hours, as happened after the
picnic.

Blood samples from the patients showed no signs of bacterial infection,

which further suggests that this was a case of intoxication. Since
intoxication is due to secreted toxins, bacteria are not usually detected in
blood or stool samples. MacConkey agar and sorbitol-MacConkey agar
plates and xylose-lysine-deoxycholate (XLD) plates were inoculated with
stool samples and did not reveal any unusually colored colonies, and no
black colonies or white colonies were observed on XLD. All lactose
fermenters on MacConkey agar also ferment sorbitol. These results ruled
out common agents of food-borne illnesses: E. coli, Salmonella spp.,
and Shigella spp.

Analysis of the chicken salad revealed an abnormal number of gram-

positive cocci arranged in clusters (Figure 1.6.81.6.8). A culture of the
gram-positive cocci releases bubbles when mixed with hydrogen

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peroxide. The culture turned mannitol salt agar yellow after a 24-hour
incubation.

All the tests point to Staphylococcus aureus as the organism that

secreted the toxin. Samples from the salad showed the presence of gram-
positive cocci bacteria in clusters. The colonies were positive for catalase.
The bacteria grew on mannitol salt agar fermenting mannitol, as shown
by the change to yellow of the medium. The pH indicator in mannitol salt
agar is phenol red, which turns to yellow when the medium is acidified
by the products of fermentation.

The toxin secreted by S. aureus is known to cause severe gastroenteritis.

The organism was probably introduced into the salad during preparation
by the food handler and multiplied while the salad was kept in the warm
ambient temperature during the speeches.

Questions
1. What are some other factors that might have contributed to rapid
growth of S. aureus in the chicken salad?
__________________________________________________________________
__________________________________________________________________

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__________________________________________________________________
__________________________________________
2. Why would S. aureus not be inhibited by the presence of salt in the
chicken salad?
__________________________________________________________________
__________________________________________________________________
__________________________________________________________________
__________________________________________

PART 2. Video Analysis

Instruction. Watch the video found in https://www.youtube.com/watch?
v=sxa46xKfIOY titled Gram Staining and summarize the staining
technique demonstrated by the scientist using the template below:

Laboratory Experiment/Process done:

_______________________________________________________________________
Materials:
_______________________________________________________________________
Organism observed:
_______________________________________________________________________
Safety equipment/procedure:
_______________________________________________________________________
Staining Technique procedure:
1.
2.
3.
4.
5.

KEY POINTS
1 Chemically defined media contain only chemically known
components.
2 Selective media favor the growth of some microorganisms while
inhibiting others.
3 Enriched media contain added essential nutrients a specific
organism needs to grow

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4 Differential media help distinguish bacteria by the color of the

colonies or the change in the medium.

REFERENCES

Centers for Disease Control and Prevention, World Health Organization. “CDC
Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria
meningitidis, Streptococcus pneumoniae, and Haemophilus influenza. WHO
Manual, 2nd edition.” 2011. http://www.cdc.gov/meningitis/lab-ma...ull-
manual.pdf

Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State

University), Anh-Hue Thi Tu (Georgia Southwestern State University),
Philip Lister (Central New Mexico Community College), and Brian M.
Forster (Saint Joseph’s University) with many contributing authors.
Original content via Openstax (CC BY 4.0; Access for free
at https://openstax.org/books/microbiology/pages/1-introduction)

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