NOTE: WE ARE ONLY DOING THE EXPERIMENTS IN THE LAB MANUAL, BUT THIS DOCUMENT IS TO TEACH YOU ABOUT ADDITIONAL EXPERIMENTS, AND THIS WILL BE ON THE LAB EXAM
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.
- 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.
- To measure how many living organisms per ml there are in asample, 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.
- 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 100% 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.
Since our original sample is in nutrient broth (a brown color), the color particles in the broth will deflect some light, 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 broth. To do this, we prepare a “blank”, which is a cuvette of sterile broth (the cuvette with a lid on it), place it in the spectrophotometer, and then set the machine to zero. That will tell the machine to ignore the color of the broth. 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 85% Transmission. That means that the color of the broth is reflecting 15% 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 + 15 = 75% 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 85% transmission, it will now read 100%. A transmission reading should be between 1-99%. The next sample is 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 transmission of light passing through a liquid sample. It is the total amount of light transmitted 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, 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.
- STANDARD PLATE COUNT (SPC)
We will take a loopful of the water sample, put it on a plate, let the bacteria grow for a few days, and count the number of colonies. 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.
We will work as one group. We will take 1ml of our original water sample (we will use a pure culture of E. coli and pretend it is a water sample) and add it to a bottle that contains 99 ml of water, and label that Bottle A. Therefore, Bottle A will contain a 1:100 dilution of the original. If we plate from this bottle, it will grow too many colonies to count. Therefore, we will take 1 ml from Bottle A and place it in Bottle B, which also contains 99 ml of water. Therefore, Bottle B will contain a 1:10,000 dilution of the original. We are not sure if Bottle B will grow too many organisms to count, so we’d better make another dilution. Add 1 ml of Bottle B to Bottle C, to make a 1:1,000,000 dilution.
PREPARING PLATE FROM BOTTLE B
Use a pipette to remove 1.0 ml from Bottle B and put it in one Petri dish. Then pour a tube of melted agar on top of it, swirl gently to mix.
PREPARING PLATE FROM BOTTLE C
Use a pipette to remove 1.0 ml from Bottle C and put it in one Petri dish. Then pour a tube of melted agar on top of it, swirl gently 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 Bottle A 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.
Bottle B = 1:10,000 800 colonies
Bottle C = 1:1,000,000 200 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 second plate (bold above) because that is the only plate in the 30-300 range.
Solution:
200 x 100,000 = 20,000,000
Covert to scientific notation and write units (org/ml)
2.0 x 107 organisms/ml
That is the number of living organisms were present in the original 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 = 50,000org/ml
5.0 x 104 org/ml
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 2 (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 or less H+ ions than pH 2 or pH 4. 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: 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 isan 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 did this experiment (we will not), you would notice that the colors in each tube are different. Some are dark yellow, light yellow, dark brown, light brown. Select the first of your samples to place into the spectrophotometer. Examine the color of your sample, then examine the colors of the series of blanks in the rack by each spectrophotometer. Select the blank that most closely matches the color of your sample. Use that blank to zero the machine, then get the transmission reading of your first sample. To get your sample into the cuvette, just pour it in carefully. Remember to make sure the reading says %T instead of Abs. Then select the blank with the color that matches your next sample, zero the machine again, and get your next transmission reading. Make sure all of your transmission readings are less than 100%. If they are not, first make sure that the machine is set on transmission (%T) instead of absorbance (Abs). If the machine is set correctly but you still have a reading greater than 100%, just record it as 100%. After you have all of your transmission readings, write them in black on the board, in the box for each pH listed. Once the entire class has written all their transmission readings on the board, we will calculate the class average for each pH tube and write the average in red. Use that red average number to calculate OD for each organism at each pH. Which organism had the highest OD for pH 2? That organism grew best in that pH, so it was an acidophile. Which had 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. Be ready to recall 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. 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 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. Remember our Serratiaorganism from the red, white, and blue colony experiment? It only makes the enzyme that turns red at 25 °C, so it produces red colonies only at room temperature. At our body temperature (38°C) it’s enzyme is denatured, so it does not turn red; it just stays white. 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 will grow cultures of bacteria at different temperatures. Next lab time, we will 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.
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