Lab 4 Instructions
Get your two P. aeruginosa plates that you streaked for isolation. Get your soil plates (PP1). Get
your plates and slants for your home experiment. Also get your chapter 12 plates: Nutrient agar
(NA), Mannitol Salt Agar (MSA: light red plate), and Eosin- Methylene Blue (EMB) agar (dark
red plate). Get the 4 demo dilution series plates. Get your red, white, blue plate if we made one.
Record the results of your last lab’s experiments in the pages at the end of Ch 4, 11 and 12.
Chapter 4: Aseptic Technique
You used a broth of Pseudomonas to Streak for Isolation on one NA (nutrient agar) plate and
you used a slant of Pseudomonas to Streak for Isolation on another NA plate. Examine both of
those plates today to see if you were able to successfully isolate a single colony in the fourth
quadrant of each plate. Then discard the plates.
Chapter 11: Standard Plate Count
Which plate is countable? What makes it countable? Estimate the number of colonies, then
calculate the colonies per ml in the original sample. Then discard the plates.
Chapter 12: Special Media for Isolating Bacteria
Each group had three Petri dishes divided into quadrants with the following organisms on each
quadrant:
1) E. coli (Gram neg rod)
2) Pseudomonas aeruginosa (Gram neg rod)
3) Micrococcus luteus (Gram + cocci that makes yellow colonies)
4) Staphylococcus epidermidis (Gram + cocci that makes white colonies)
a) Nutrient agar: How many quadrants had growth? Why?
b) Mannitol Salt Agar (light red plate): How many quadrants had growth? Why did some
not grow? Which colonies were white and which were yellow? Did any of the quadrants
turn the MEDIA yellow? What organism would turn the media yellow? What causes the
media to turn yellow? The yellow color in the media will differentiate the species of
Staphylococcus. Staph aureus and Staph saprophyticus (yellow colonies) are mannitol
fermenters, while Staph epididymis and Micrococcus (white colonies) do not ferment mannitol.
To determine the difference between the two yellow colonies that ferment mannitol (Staph aureus
and Staph saprophyticus), we could do a coagulase test (we will not), S. aureus would be the only
positive organism. To tell the difference between the two white colonies (Staph epididymis and
Micrococcus), we would place a Novobiocin antibiotic disc and only Staph epididymis would
show a zone of clearing around it.
c) Eosin- Methylene Blue (EMB) agar (dark red plate): How many quadrants had growth?
Why?
d) Discard the plates when you are done.
1
SOIL PROJECT
Choose the best antibiotic-secreting colony and make your working stock:
1) Check your ESKAPE plate for zones of inhibition.
2) Take a picture of your Test Plate with the zones of inhibition. Make sure you get
a good photo of the zones on the plate.
3) Choose the colony with the biggest zone, and streak for isolation on a new TSA
or LB plate (pick the same agar that your organism is on).
4) Next time, use a needle to take an isolated colony from your streak for isolation
plate and streak it on two slants.
5) Keep your Master Plate as a working stock and use it to run all your
biochemical tests.
6) You can now discard your original plate from Lab 1.
7) Put the Master Plate back in the refrigerator, upside down.
8) The new streak for isolation plate will stay upside down at room temperature for
the weekend before it goes into the refrigerator.
9) Observe your colonies with a zone of inhibition and record their morphological
characteristics as indicated below.
1. MARGINS (look at the surface of the plate)
a. Filiform (thread-like edge)
b. Arborescent (tree-like branches)
c. Beaded (starts smooth, breaks into individual
colonies at top)
d. Effuse (edges are not clearly defined)
e. Rhizoid (root-like branches)
f. Echinulate (serrated edge)
2. TEXTURE (look at surface of plate)
g. Flay, dry (Bacillus megatarium)
h. Spreading (proteus, pseudomonas)
i. Crusty, waxy (mycobacterium)
j. Transparent
3. PIGMENTATION (color of plate)
k. Yellow
l. White
m. Green
n. Etc
4. ELEVATION
o. Convex
p. Flat
q. Raised
r. Umbonate
Pre-Lab Lecture
Total plates to keep per student
for the duration of the
experiment:
1) Master Plate (original with
grid lines)
2) Test Plate with grid lines.
3) Isolation plate of the one
organism that has the best
zone of inhibition from the
Test Plate
EVALUATION OF ANTISEPTICS AND DISINFECTANTS
An antiseptic is used on tissue, not inanimate objects. There are two kinds of antiseptics: topical
(like Bactine) and oral (like Scope and Listerine). Disinfectants are chemicals that are used on
inanimate objects only.
2
Sterile paper discs can be soaked in different disinfectants or antiseptics and then placed on a plate
that has been grown with a particular organism. In a few days, the clear zone around each disc can
be measured. For antiseptics and disinfectants, the products with the largest zones of inhibition
are the ones that are the most effective. Note that this is NOT the case with antimicrobials,
where the largest zone is not necessarily the best.
HAND SCRUBBING EXPERIMENT
A BACTERIOSTATIC agent is one that causes temporary inhibition of growth of bacteria.
A BACTERIOCIDAL agent is one that kills bacteria.
An ANTISEPTIC is a chemicals used on living tissue to decrease the number of microbes
A DISINFECTANT is a chemical used on NON-living tissue to decrease the number of microbes
A nosocomial infection is one that is acquired after a patient is admitted to a hospital. Nosocomial
infections can be avoided by proper hand washing between patients.
Transient organisms are those which are on our hands and arms for a short while. We pick these up
by touching objects, etc. Washing with soap and water, even for a short time, will remove some
transient organisms. Resident organisms are those which stay on us, even after we wash with soap
and water. Our resident organisms do not cause us disease unless there is a break in the skin.
However, your resident organisms might be harmful to another person. Washing your hands with
plain water rinses off a few transient organisms. Washing with soap and water removes many more
transient organisms and some resident organisms. Resident organisms cannot be completely
removed from the skin; it is impossible to sterilize living tissues.
INSTRUCTIONS FOR TODAY’S LAB
Chapter 24: Chemical Methods of Control, Disinfectants and Antiseptics
Each lab group on the right side of the room will use
1) Pseudomonas aeruginosa
Each lab group on the left side of the room will use
2) Staphylococcus aureus
Each lab group will have Mouthwash, Pine-sol, and contact lens cleaning solution in a tube.
Each lab group will also have three Petri dishes with nutrient agar (NA).
Use your wax pencil to divide all three plates into 5 portions, as seen on p. 186 (below fig) and label them
0, 2.5, 5, 10, and 20 (ignore the picture on the right side in the lab manual; all three plates will be marked
according to the picture on the left). The numbers represent the number of minutes that the organism has
been exposed to the disinfectant. Also label the bottom edge of your plate with the name of your
disinfectant, your name, date, and Magrann.
Each of three people in your lab group needs to select one disinfectant and one plate to be in charge of.
Then add 0.5 ml of your bacteria to your disinfectant tube and spank the bottom of the tube with your
knuckle to mix, and start the timer. Immediately take a sterile swab of bacteria and streak the zero
quadrant of your plate. Wait 2.5 minutes and take another swab to inoculate the 2.5 quadrant of the same
3
plate. Repeat for the 5, 10, and 20 minute quadrants. Put the dishes with the agar facing up in the
incubator.
In the next lab period, we will see how many minutes it took each product to kill all the bacteria. Note the
active ingredient of each disinfectant on the bottle’s label, and write this on p. 188 question 2.
Chapter 25 (p. 191)
Summery
Each lab group takes 3 Petri plates of Mueller-Hinton agar (looks like nutrient agar).
Inoculate one plate with Staph aureus (Gram +), one with E. coli, and one with Pseudomonas aeruginosa
(both Gram neg) by streaking in complete coverage in a tight zig-zag throughout the plate, using a sterile
swab (called streaking for confluence). Then take 5 different antibiotic discs and set them on each plate
(ID number of the disc facing up so you can read it; see p. 192 for photo) and measure the zone of
inhibition next lab period.
Details
Make sure you use the Mueller-Hinton agar. You do not need to draw quadrants on these plates.
Take three Mueller-Hinton agar plates and label each dish on the bottom with the organism, date, group
leader name, and Magrann.
To keep a cotton swab sterile when removing it from the package, use one thumb and index finger to
grasp each side of the proper end of the package (the wooden stick end). Roll your thumbs away from
each other to allow the swab handle to pop out of the paper. Do not let the swab touch the outside of the
paper package or it will no longer be sterile. Leave the cotton part in the package until you are ready to
use it.
Dip a sterile swab into the culture, express the excess liquid from the swab by pressing it against the
inside of the tube. Use one hand to lift the lid of the Petri dish just enough to inoculate with the other
hand. Do not remove the lid and set it on the desk! Streak EVERY MILIMETER of the whole plate in a
TIGHT back and forth zig-zag motion, and discard the swab into the biohazard bin. Return your culture to
the original rack in the back. Do likewise with the other two cultures.
There is a bag with 9 different types of antibiotic discs. Choose 5 of them to use on each of your three
plates. When you reach in the bag to obtain a tube of antibiotic discs, keep your fingers away from the
ends of the tubes. Withdraw the tube and look at both ends and notice that one end has the antibiotic disc
exposed. If you squeeze the metal clip on the tube, it will push against the first disc so that it sticks out
from the tube and you can grab it with forceps. Let the disc fall onto the dish in the desired position, then
gently tap the top of the disc with the forceps to make sure it sticks. See page 192 for a photo of
placement of the discs. They should be in a clock-formation, approximately equal distances from each
other. After your discs are placed, go to page 195 in your lab manual and write down the name of your
antibiotics and their code numbers that are printed on the disc (see page 193 for code numbers and name).
The dishes go in the incubator, agar side up.
4