Lab 3 Instructions
Finish the lab from last week: Look at your slides from last week if you did not finish.
Go get your cultures from last lab: 3 slides in your drawer, 2 broths, 2 slants, your pick and
patch soil plate (label them Master Plate PP1).
Chapter 4: Aseptic Technique….Streak for Isolation
Use the slant of Pseudomonas aeruginosa that you inoculated last week and streak for isolation
onto a nutrient agar plate. See page 91 in your lab manual or your Lab Unit 1 instructions. Be
sure to flame your loop after you inoculate the first quadrant, wait for the loop to cool, then drag
your loop ONCE across quadrant 1(through the middle) and steak it into quadrant 2. Flame your
loop and drag it ONCE across quadrant 2 and streak it into quadrant 3. Flame your loop and
repeat for quadrant 4. Turn your plate counter-clockwise after you perform each streak. Keep the
zig-zags close together as you streak.
Then use the broth of Pseudomonas aeruginosa that you inoculated last week and streak for
isolation another nutrient agar plate in the same way. Place both plates in the incubator and
check it next time to see if you were able to successfully isolate a single colony in the fourth
quadrant of each plate.
Chapter 12: Special Media for Isolating Bacteria
Each group should take one of each of these Petri dishes:
Nutrient agar (NA)
Mannitol Salt Agar (MSA: light red plate)
Eosin- Methylene Blue (EMB) agar (dark red plate)
Put your name, date, Magrann, and “Ex 12”. Use your wax pencil to mark 4 quadrants onto
each of the three plates. Label each quadrant with 1-E, 2-P, 3-M, or 4-S to correspond to the four
organisms below. Each plate will have one organism per quadrant. Flame the loop for the first
organism and inoculate all three plates; you do not have to re-flame between the plates. Flame
the loop between the organisms. Use a loose streak pattern for each quadrant. See p. 98 of
manual. When you return your pure culture of the organisms in the tube, do NOT tighten the
lids all the way or the colony will die out for the next class. The cap should be loosened by a half
of a turn.
We will work with 4 organisms for this experiment:
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)
The MSA plate contains a lot of salt. Only Gram positive organisms can tolerate this, so which
of your quadrants should have growth next time?
The Eosin and Methylene Blue plate contains Eosin and Methylene Blue. They are dyes that
inhibit Gram positive bacteria, but are able to grow Gram negative bacteria. Which of your
quadrants should have growth next time?
1
MSA and EMB agar are therefore SELECTIVE MEDIA because only certain types of bacteria
will grow on them. All of your organisms will grow on the Nutrient agar, so it is not selective.
MSA also contains a sugar called mannitol. Only certain organisms will use the sugar. If the
sugar is used, the waste product of its metabolism is an acid. The red color in the plate is from
methyl red, a pH indicator that will turn yellow if acids are present. If the media turns yellow
next week, the organism fermented the mannitol sugar. You should have two organisms that
survive on this agar next week, and one is a mannitol fermenter (media will turn yellow in that
quadrant) and the other is not (media will stay orange-pink). This demonstrates that we can grow
more than one organism on that plate and see that one ferments mannitol and the other does not.
That means that this media is also DIFFERENTIAL media. We can differentiate between two
organisms growing on one plate. Therefore, MSA is selective AND differential media, while
EMB is just a selective media. Salt makes MSA selective, and mannitol makes it differential.
When E. coli is grown on EMB agar, it will have a green metallic sheen as it absorbs the dyes. It
is the only organism that does that. Look for that next lab period.
Another type of media is ENRICHED media, such as blood agar. In this media, sheep’s blood is
added to nutrient agar. This plate would grow organisms that are fastidious (picky eaters), such
as pathogenic bacteria like Streptococcus pyogenes (causes strep throat). With this media we can
also see whether the organism demonstrates hemolysis (has an enzyme that ruptures red blood
cells). There are three types of hemolysis. Alpha hemolysis (blood turns green from partial
breakdown of the iron in the RBC), beta hemolysis (blood agar turns clear, from complete
breakdown of the RBC), or gamma hemolysis (no change in blood color because the organism
does not make any enzymes that break down RBCs).
SOIL PROJECT
Find the colonies that secrete antibiotics
1) Select 2 broths of a pure culture of bacteria from the back. Make sure one is a Gram
positive and one is a Gram negative.
2) Choose your best looking plate and note what type of agar it is (usually TSA). Get 2 new
plates of that same kind of agar; one will be streaked for confluence with your Gram
positive pure culture, using a cotton swab. The other will likewise be streaked with the
Gram negative.
3) Get 6 cotton swabs
4) Get a paper grid (supplied in lab),
5) Label one plate as Gram Positive PP2, organism name, Soil, Magrann, date, your name,
and the name of the organism that you will streak the plate with. Label the other plate the
same except call it Gram Neg PP2, and the organism name will also be different.
6) Then use the paper grid and draw it onto the bottom of three new plates and number the
squares with the number of different colonies you have. IGNORE ANY OF YOUR
COLONIES WITH A RIZOID MARGIN…THOSE ARE FUNGI AND WE ONLY
WANT BACTERIA.
7) Streak for confluence on your 2 new plates. To do this, get a cotton swab, dip it in your
organism broth. Swab your new plate from top to bottom without leaving any gaps.
Discard the swab in the biohazard bin
8) Use a NEEDLE to transfer colony #1 from your old MASTER Plate PP1 to grid square
#1 of your new Gram positive PP2, and repeat for all the colonies. Skip the numbers on
2
colonies that you are not transferring. For example, if organism #4 is a fungus on your
old plate, do not number your new plate with any #4. The numbers of the organisms on
your first plate will be like their name. If your plate is overgrown with fungus, discard it
and do not use it anymore.
9) Incubate upside down at room temperature. The Tech will put them in the refrigerator on
Monday so they do not overgrow.
Your organism is called an ESKAPE pathogen.
ESKAPE pathogens are a group of bacteria with a high rate of antibiotic resistance that are
responsible for the majority of nosocomial infections.
 Enterococcus faecium
 Staphylococcus aureus
 Klebsiella pneumoniae
 Acinetobacter baumannii
 Pseudomonas aeruginosa
 Enterobacter species
However, Yale does not want us to work with the above dangerous organisms, so we will use these close
relatives instead because they are safer for students to work with. One of your plates should have one of
these Gram negatives, and the other plate should have one of these Gram positives.
Gram Negative
Staphylococcus epidermidis
Staphylococcus cohnii
Bacillus subtilis
Gram Positive
Staphylococcus epidermidis
Staphylococcus cohnii
Bacillus subtilis
Chapter 11:
ENUMERATION OF BACTERIA
Sometimes, an emergency room contacts the Center for Disease Control (CDC) to let them know that
they had an unusual amount of people come in with the same illness. Let’s say there is a sudden outbreak
of the stomach flu. The CDC interviews each patient to find if they all went to the same location right
before they got sick. Let’s say that these patients all went to the same public park. The CDC then
examines the park and makes a list of all the possible places that organisms might be, such as a food
vendor, a lake, picnic tables, water fountains, and bathrooms. They then ask the patients to tell them
everything they touched while at the park. Let’s say the CDC found that everyone who got sick drank out
of the same water fountain. They think the fountain might be contaminated by something, perhaps a
broken sewer line underground. They send a sample of the water to the lab, and they want to know how
many organisms per ml there are. Since drinking water is not sterile, there are guidelines as to how many
bacteria are within acceptable limits. We need to determine how many organisms are present in the
sample. We need to tell the CDC how many living organisms are present in the water, and how many
living plus dead organisms are in the sample.
1. To measure how many living and dead organisms per ml there are in the sample, we
measure the turbidity (cloudiness) by placing it in a machine called a spectrophotometer.
This is considered to be an indirect method of enumerating bacteria (aka estimating cell
density), and relies on the cloudiness of the sample.
3
2. To measure how many living organisms per ml there are in a sample, we plate them and
count the colonies that grow. This is called a Standard Plate Count (SPC). This is
considered to be a direct method of enumeration of bacteria. Before we can perform a
SPC, we need to perform a serial dilution of the original broth culture. Otherwise, we
will grow too many organisms to count.
1. TURBIDITY MEASUREMENT
To measure turbidity, we use a spectrophotometer. You need to understand how this
machine works. This machine sends a beam of light through a tube of liquid sample, and
reads how much light comes out. If we send a beam of light through a tube of clear water, we
will get 100% of light out, and the machine will read 100% transmission. If our sample is not
clear because it contains organisms, we will get something less than 100% transmission. The
light that is not transmitted is absorbed. If 80% of light is transmitted, 20% is absorbed.
Transmission + Absorption = 100%. Therefore, transmission and absorbance are inversely
related. Samples are placed in special tubes called cuvettes, which are test tubes of
optically pure glass that will not absorb light like regular glass test tubes. These are placed
into the spectrophotometer, and the transmission is read for each sample.
Turbidity Measurement Procedure
Start with a 150 ml flask (1) of water inoculated with 10 ml E. coli. This will represent the
water we got from the water fountain at the park from the CDC. Perform a serial dilution as
follows:
1. Remove 1 ml of water and add it to 99 ml of sterile water in a flask (2). Shake.
2. Remove 1 ml of water from flask (2) and add it to 99 ml of sterile water in a flask (3) and
shake.
3. Remove 1 ml of water from flask (3) and add it to 99 ml of sterile water in a flask (4) and
shake.
4. Place a cuvette of sterile water in the spectrophotometer and set the machine to zero.
5. Place a cuvette of the water from flasks 1- 4 (one at a time) and measure the transmission
reading.
Since our original sample may have some color from the water, the color particles in the
water will deflect some light, falsely lowering the light transmission. We need to remove that
factor so we can determine only how much light is being deflected by the organisms in the
tube. To do this, we prepare a “blank”, which is a cuvette of sterile water, placed in the
spectrophotometer, and then set the machine to zero. That will tell the machine to ignore the
color of the water. How does putting the blank in and zeroing the machine work? If we
put the blank in but do not set the machine to zero, the machine will not know that this is our
“blank”, and it would give us a transmission reading, let’s say 95% Transmission. That
means that the color of the water is reflecting 5% of the light. If we then put our sample in,
the transmission of our first sample might be 60% Transmission. However, the correct
transmission of our sample should have been 60 + 5 = 65% transmission. Without using the
blank and setting the machine to zero, our readings will be lower than they should be.
When we put the blank in and set the machine to zero, instead of reading 95% transmission,
it will now read 100%. A transmission reading should be between 1-99%. The next sample is
4
placed in the machine (it does not need to be blanked and zeroed anymore), the reading is
obtained, and likewise for all the samples.
Once you have your transmission readings, turbidity is recorded as a sample’s optical
density. Optical Density (OD) is a measurement of the absorbance of light passing
through a liquid sample. It is the total amount of light absorbed in a sample that
contains both living and dead bacteria. Notice that the cloudier (more turbid) our
sample is, the lower the transmission (inversely related to OD). Low transmission (and
high OD) means there are a lot of bacteria in the sample. High transmission (and low
OD) means there are few bacteria in the sample. Therefore, OD and transmission are
inversely related. We don’t report the transmission, just the optical density.
When the CDC sees our preliminary report that the water sample from the drinking fountain
has a high optical density, (low transmission, so there are a lot of organisms) they will be
suspicious that this is the source of the outbreak of stomach flu. A high optical density
indicates that there are many particles in the water, but it might not be a dangerous situation,
because the particles might be harmless mineral deposits. To find out if the cloudy water is
from living organisms, we will have to grow them on a plate and count the number of
colonies. Why didn’t we just do this in the first place? We can do the turbidity test in a few
minutes to give the CDC a quick screen test. The plate count takes a few days to grow.
2. STANDARD PLATE COUNT (SPC)
To count the number of colonies on a plate, we need to have a plate that does not have too many
colonies to count. To make such a plate, we need to dilute the original first. Since we don’t know
how much to dilute it, we do a series of dilutions (a dilution series), then plate each dilution we
make, and see which plate grows the number of colonies that are within our ability to count. The
number of colonies that are within our ability to count are 30-300. Therefore, we will plate a
dilution series, let the bacteria grow for a few days, and then see which plate has 30-300
colonies. That will be the only plate we will count.
5
Flask 2 will contain a 1:100 dilution of the original. If we plate from this bottle, it will grow too
many colonies to count. Flask 3 will contain a 1:10,000 dilution of the original. We are not sure
if Flask 3 will grow too many organisms to count, so we’d better make another dilution. Flask 4,
will be a 1:1,000,000 dilution.
PREPARING PLATE FROM Flask 3
Use a pipette to remove 1.0 ml from Flask 3 and put it into an empty sterile Petri dish. Then pour
a tube of melted agar on top of it, swirl gently to mix. Then use a micropipette to remove 0.1 ml
from flask 3 and put it into another empty sterile Petri dish. Pour a tube of melted agar on top
and swirl to mix.
PREPARING PLATE FROM Flask 4
Use a pipette to remove 1.0 ml from Flask 4 and put it into an empty sterile Petri dish. Then pour
a tube of melted agar on top of it, swirl gently to mix. Then use a micropipette to remove 0.1 ml
from flask 4 and put it into another empty sterile Petri dish. Pour a tube of melted agar on top
and swirl to mix.
We will then let the plates grow for a few days to see which plate has 30-300 colonies, and we
will count them and calculate the number of organisms per ml in the original solution.
Each dilution in our series is a one hundred fold (1:100) dilution, but compared to the original,
the last dilution will be one to a million (1:1,000,000). We already know Flask 2 will not be
dilute enough, so there will be too many colonies to count, so we don’t plate from the first
dilution. Let’s say the below list is how many colonies we count on what was plated from the
dilution series. Which of the below plates will we use to count colonies? Discard the other
plate.
Flask 2 = 1:100  800 colonies
Flask 3 = 1:10,000  200 colonies
Flask 4 = 1:1,000,000  20 colonies
Counting is only significant if the colony count is 30-300 colonies per plate. If there are more
than 300, some colonies may overgrow on other colonies, and the count is not accurate. If there
are less than 30, there are not enough to count. Which of your plates is in that range? Use ONLY
that plate to calculate organisms per ml.
CALCULATE THE NUMBER OF LIVING ORGANISMS IN A SAMPLE
After you count the number of colonies in your appropriate plate, we need to multiply that
number by the dilution factor to see how many organisms were in the original.
FORMULA:
Concentration = Colony numbers x Dilution Factor
Which plate do you use to apply this formula?
We have to use Flask 3 because that is the only plate in the 30-300 range.
6
Solution:
200 x 10,000 = 2,000,000
Covert to scientific notation and write units (org/ml)
2.0 x 106 organisms/ml
That is the number of living organisms were present in the original sample.
Sample Problem:
We counted 50 colonies on a plate that has 1:1000 dilution. What is the total number of
organisms in the original solution?
Solution:
50 x 1000 = 50,000 org/ml
5.0 x 104 org/ml
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
Remember to leave your plates upside down during the week, so the agar is on the top.
Why do we do this? It prevents condensation from developing on the inside lid, and then
dropping onto your agar, mixing the organisms. It also makes it easier to retrieve the plate from
the sleeve container. If you do not put the plate in upside down, and you try to lift it, you might
accidentally lift the lid off your dish, exposing the agar to the air and contaminating it. Keeping
the plates stored upside down also enables you to read the important writing on the bottom of the
plate. After you examine your plate next week, select the very best looking white colony which
is separated from the other colonies, touch it with a sterile needle, and zig-zag it across the
surface of a nutrient agar slant. Do the same for each of the other two color colonies, so you end
up with three slants, one of each organism. You will then let those grow until the next lab period.
You will know if your isolation technique was successful if there is only one color in each tube.
Remember that the organisms for this experiment need to stay at room temperature.
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
NOTE: WE ARE NOT DOING THE FOLLOWING THREE EXPERIMENTS BUT
THIS MATERIAL IS STILL ON THE LAB EXAM
EFFECT OF pH ON GROWTH OF BACTERIA
pH measures the concentration of hydrogen ions (H+). The more H+ ions, the more acidic
the substance is, which means it has a low pH (below 7). The less H+ ions, the more basic
(or alkaline) the substance is, which means it has a high pH (above 7). Substances with
neutral pH (like water and much of our body fluids) are pH 7.
The pH scale runs from pH 1 (acid) to pH 14 (base). Each unit change in pH represents a
10 fold difference in H+ ions. That means that pH 3 has 10x more H+ ions than pH 2.
Since acids have more H+ ions, a change from pH 3 to pH 4 represents a 10x decrease in
H+ ions. A change from pH 3 to pH 2 represents a 10x increase in H+ ions. A change
from pH 3 to pH 5 represents a 100x decrease in H+ ions. A change from pH 3 to pH 6
represents a 1000x decrease in H+ ions. What is the difference in going from pH 10 to pH
2? Answer: Subtract 2 from 10 and get 8. That is the number of zeros to place after the one:
7
100,000,000. Then say whether it is an increase or decrease. If you go from a high pH to a
low pH, it is an increase in H+ ions. Therefore, going from pH 10 to pH 2 represents a
100,000,000 increase in H+ ions. Likewise, going from pH 2to pH 10 represents a
100,000,000 decrease in H+ ions.
Some organisms prefer a particular pH. If they are placed in a substance that is suboptimal
(meaning less than the best for them), the proteins in their cell walls of the vegetative cells
can become denatured (damaged).
Organisms that grow best at pH 2- 4 are acidophiles
Organisms that grow best at pH 7 are neutrilophiles
Organisms that grow best at pH 10-12 are alkaliniphiles
Suppose we use the below organisms to inoculate a series of pH tubes, let them grow for a few
days, and use the spectrophotometer to measure growth. We could then calculate their optical
density. The tubes with the highest OD (and the lowest transmission readings) contain the most
bacteria.
Tube 1 has Alcaligenes faecalis
Tube 2 has Saccharomyces cerevisiae
Tube 3 has Staph aureus
Alcaligenes faecalis is an opportunistic pathogen, meaning that it only causes infection if it
has an opportunity to invade. This organism causes urinary bladder infections, so a patient
with a catheter might be at risk; the catheter provides the opportunity for the organism to get
in. This bacterium degrades urea to make ammonia, which increases the pH. Therefore, this
organism is an alkaliniphile.
Saccharomyces cerevisiae is a yeast which is used to make beer. Many yeasts and fungi use
fermentation as a metabolic pathway, and acids are produced in the process. This organism
is an acidophile.
Staph aureus is a resident organism (lives on our skin without causing disease) which is also an
opportunistic pathogen. If we get a cut, it can take the opportunity to invade and cause the wound
to become infected. It is a neutrophile.
If you inoculate each organism into three tubes (acid, base, and neutral), they would grow the best
at the pH they prefer. Which organism would have the highest OD for pH 2? That organism
grew best in that pH, so it was an acidophile. Which would have the highest OD for pH 12?
That organism grew the best in that pH, so it was an alkalinophile. Continue this pattern for
all the pH tubes for all three organisms. Know which organism was an acidophile, which was a
neutrophile, and which was an alkainophile.
EFFECT OF UV LIGHT ON GROWTH OF BACTERIA
What is effect of ultraviolet (UV) light on different types of bacteria? The DNA of bacteria
contains two adjacent thiamine’s (a nucleic acid). UV light causes a bond to form between the
two thiamine’s, and this stops DNA replication. This is the cause of death in the organism. If
8
an organism can produce endospores, it could survive chemicals, heat, and UV light
exposure for a longer time. You could design an experiment to expose plates of two organisms
to UV light for various periods to see how long it takes to kill each type of bacteria with UV light.
Gram positive rods are the only bacteria that make endospores. An example is Bacillus.
Staph aureus (Gram positive cocci) is exposed to a various number of seconds of UV light
Bacillis (Gram positive rod) is exposed to a various number of minutes of light. Why are we
exposing this organism to UV light for a longer period? Bacillus makes endospores, so it survives
in these conditions longer.
FORMULA TO MEASURE RESISTANCE TO UV LIGHT
Time to kill Bacillus ÷ time needed to kill Staph.
Note: to apply this formula to another two organisms, Divide the longest time by the shortest
time. Suppose the more resistant organism took 30 minutes to kill, and the other organism took 10
minutes to kill. 30 ÷ 10 = 3. That means organism 1 is 3x more resistant to UV light than
organism 2. It also means that organism 2 is 1/3 as resistant to UV light than organism 1.
TEMPERATURE REQUIREMENTS OF BACTERIA
Sometimes we incubate plates in a heater or cooler, and sometimes we leave them in room
temperature. Why? Organisms have different temperature requirements. Human
pathogenic organisms prefer our body temperature (37 °C). Non-pathogenic organisms
might prefer room temperature (25 °C). The type of pathogenic bacteria that have flagella
for motility often exist in two different forms: they have a flagella when they are outside of
a human body, and they lose their flagella when they enter the body. Remember, when we
are testing for motility of an organism, we have to incubate at room temperature, or they
will lose their flagella and test as non-motile. Organisms have various temperature
requirements in order to have maximum enzyme activity. Overall, bacteria are more heat
resistant than most other forms of life, but they can only tolerate a certain amount of heat.
Generally, if heat is applied, microbes are killed; if cold temperatures are used, microbial
growth is inhibited.
Psychrophiles: like freezing cold temperatures
Mesophiles: prefer anything from room temperature to body temperature (25-40 °C).
Thermophiles like it quite hot (45-65 °C)
Hyperthermophiles: like extreme heat, such as found in volcanoes.
Most human pathogenic bacteria are mesophiles. We could grow cultures of bacteria at
different temperatures and at the following lab time, record their transmission readings in
the spectrophotometer and calculate their optical density. Those with the highest OD are
the ones that grew the best at that temperature.
9