Mrs. K. Willner
Health Sci 20
HS 20 – MD1: Examine the operating principles of various medical diagnostic tools and
procedures including the advantages and disadvantages of each diagnostic tool.
c. Explain the procedures and relevance of various laboratory tests (e.g., blood testing,
culture swabs, urinalysis, biopsy, and microscopy) in medical diagnosis
Also possible connections with HS 20 – HB2: Examine a variety of pathologies (pathological
concerns, pathological issues) and how they affect the body.
Culture Swabs
Purpose: This activity is based on the learning context of practicing skills and specifically, for
this activity, the skills of performing and recording.
This will take parts of several classes; as you are working on this you will also be doing some
other learning about this Unit or Outcome.
What to do:
Each step below will have to be accomplished in different days and should take only part of a 60
min period.
Step 1: Setting up the Lab
You will need to make the culture plates (medium in which the microorganisms may grow).
Follow the directions below which are adapted from the following website
http://www.sciencestuff.com/nav/instructions/agar1.htm (by the way – agar is tricky to mix, so be
sure to follow the directions to a T)
Nutrient Agar Powder
Preparation & Equipment Use
Background Information:
Agar is pronounced awger (sounds like fogger without the f). Agar is a gelling
agent extracted from red seaweed. Nutrient agar is a commonly used food medium
for microbial cultures. Nutrient agar contains:
o
o
o
o
beef extract (provides carbohydrates, nitrogen, vitamins, salts)
peptone (helps control pH)
agar (a carbohydrate used as a solidifying agent)
distilled water (an agent for distributing food materials to growing colonies
of micro-organisms)
Materials:
Agar Powder
distilled water
flask or beaker
glass stir rod
lab thermometer
sterile petri dishes (plastic)
flame for boiling mixture
heat resistant hand protection
Note: Keep sterile petri dishes closed until ready to pour agar into them.
Air-borne contaminants can easily invade an open petri dish.
Procedure:
1. Measure agar and distilled water into clean flask or beaker.
Recipe: Agar + Distilled Water = Yield
Agar + Distilled = Yield
23 g
Water
50
11.5
1000 ml plates
g
500 ml
25
9.2 g
400 ml plates
4.6 g
200 ml
20
plates
10
plates
2. Flame sterilize a clean glass stir rod to stir the medium as it melts.
3. While wearing heat resistant hand protection, hold the flask or beaker over the
flame. Swish or stir the mixture constantly while heating.
4. Boil the mixture for 1 minute. Remove from heat.
5. Place a sterile lab thermometer. in the mixture and monitor the temperature until it
falls to approximately 45 - 50° C or if a lab thermometer is not available, cover and
let stand a few minutes.
6. Pour enough melted agar into each sterile plastic petri dish to cover the bottom about 1/8" to 1/4" deep. Replace the lid immediately.
7. Place agar plates on a counter top to cool and set. Agar medium will set like stiff
gelatin at room temperature.
8. The agar medium is now ready for storage or use.
Storage: Stack agar plates upside down (so moisture can’t drip onto the agar) in the
refrigerator. Do Not Freeze! The purpose of placing the plates upside down is to prevent
condensation from dripping down onto the agar surface which could then facilitate
movement of organisms between colonies.
Step 2: Doing a Swab (Preparing the plates)
At least two plates should be swabbed per person to hopefully ensure success in growing a
colony.
1. If plates have been refrigerated, set them out and allow them to warm to room
temperature.
2. Obtain two clean cotton swabs (also known as Q-Tips) and do not touch the cotton
(trying to keep it as sterile as possible).
3. Swab a surface by rubbing the swab over it several times. If the surface is moist, like a
tongue there is no need to moisten the cotton; if it is dry then moisten the swab with
sterile water and then swab the surface.
4. Uncover each agar plate just long enough to inoculate the medium. Hold the petri dish
lid directly over the petri dish (or tilt the lid just enough to allow the cotton swab inside)
while inoculating the medium to help prevent contamination from air-borne particles.
Inoculate the medium (agar) by lightly rubbing (glide) the cotton swab on the half of the
surface (without digging in our gouging the agar) in a distinguishable pattern (such as a Z
or N) to help determine that what is growing is what you put there and not a another
contaminant.
5. Repeat step 3 on the other side of the plate.
6. Place the lid on and label with masking tape where the swabs are from.
7. Place the plate in a warm (warmer than room temp. but not sauna hot) and it should
not be in a bright location or in direct sunlight.
Incubation:
8. Turn the plates upside down (so moisture can’t drip onto the agar) and put them in a
warm place. The ideal temperature for incubation is 32° C or 90° F. Microbial growth
should start to become visible in about 2 -3 days.
Step 3: Analyzing and Recording
The following is pertinent information for several websites. There are different descriptions of
distinguishing what is growing in a culture along with the protocol for how to describe the
colonies growing in the plates.
Do the following activities to record and analyze what you have grown. (Some may not have
worked, in those cases students could join another or pair up with another group).
1. Apply the following information and determine what is growing in your petri dish.
2. Write a description of each of the different colonies you have cultured in your petri dish.
Use the Sinorhizobium meliloti as an example of how to do a well-written description.
Question:
When observing and comparing bacterial growth with fungal growth which have been
cultured in a petri dish, in general, in what ways do they differ?
Replies:
You cannot always tell just by looking. Many bacteria and fungal cells will form nice
small colonies with smooth round edges. Some fungal cells, for example, molds, will
form colonies that are "hairy", just like the mold you see on old bread. Of course, there
are filamentous bacteria too, but if I were betting the odds, I would bet a fuzzy colony
was fungal. Of course, if you have access to a microscope, the bacteria cells will not
have a nucleus, and the fungal cell will.
Paul Mahoney, Ph.D.
Most bacteria produce round (though sometimes flat, wet-like) colonies, whereas fungi
produce colonies of irregular shape, often star-like or 'hairy'. This is a rough way of
distinguishing the two.
Trudy Wassenaar
Source: http://www.newton.dep.anl.gov/askasci/mole00/mole00215.htm
Although bacterial and fungi colonies have many characteristics and some can be rare, there are a few
basic elements that you can identify for all colonies:(1)

Form - What is the basic shape of the colony? For example, circular, filamentous, etc.

Elevation - What is the cross sectional shape of the colony? Turn the Petri dish on end.

Margin - What is the magnified shape of the edge of the colony?

Surface - How does the surface of the colony appear? For example, smooth, glistening, rough,
dull (opposite of glistening), rugose (wrinkled), etc.

Opacity - For example, transparent (clear), opaque, translucent (almost clear, but distorted vision,
like looking through frosted glass), iridescent (changing colors in reflected light), etc.

Chromogenesis (pigmentation) - For example, white, buff, red, purple, etc.
Please note that 3 additional elements of morphology should be examined only in a supervised laboratory
setting: consistency, emulsifiability, and odor.
Refer to the diagram below for illustrated examples of form, elevation, and margin:(2)
What Can Grow on a Nutrient Agar Plate?
Bacteria: Each distinct circular colony should represent an individual bacterial cell or group that has
divided repeatedly. Being kept in one place, the resulting cells have accumulated to form a visible patch.
Most bacterial colonies appear white, cream, or yellow in color, and fairly circular in shape.
Yeasts: Yeast colonies generally look similar to bacterial colonies. Some species, such as Candida, can
grow as white patches with a glossy surface.
Molds: Molds are actually fungi, and they often appear whitish grey, with fuzzy edges. They usually turn
into a different color, from the center outwards.
Other Fungi: Moss green colonies, a white cloud, or a ring of spores can be attributed to the growth of
Aspergillus, which is common in such fungal infections as athlete's foot.
Source: http://www.sciencebuddies.org/science-fair
projects/project_ideas/MicroBio_Interpreting_Plates.shtml
RECIPES AND PROTOCOL
Cultures of microorganisms can be grown on any medium that is appropriate for
their isolation and cultivation. Since morphology is influenced by medium type and
growth conditions, care should be taken to record these factors. Smibert and Krieg
(5) proposed the following protocol:
1. Measure the colony diameter in millimeters.
2. Describe the pigmentation (distinguishing between pigmented colonies and those
secreting diffusible pigments).
3. Describe the form, elevation, and margin as indicated in Fig. 1. Also indicate
whether the colonies are smooth (shiny glistening surface), rough (dull, bumpy,
granular, or matte surface), or mucoid (slimy or gummy appearance).
4. Record the opacity of the colonies (transparent, translucent, or opaque) and their
texture when tested with a needle: butyrous (buttery texture), viscous (gummy),
or dry (brittle or powdery).
A well-written description would include, therefore, each of the parts of Smibert and
Krieg’s protocol. A description of Sinorhizobium meliloti is used as an example.
Ex.: “Colonies of Sinorhizobium meliloti grown on trypticase soy agar are
approximately 1 to 2 mm in diameter. They lack pigmentation and are translucent.
Colonies are smooth and are circular in form with an entire margin. They have a
convex elevation. When manipulated with a needle, the colonies are viscous.”
Source : http://www.microbelibrary.org/component/resource/laboratory-test/3136-colonymorphology-protocol