Chapter 4: Aseptic Technique .Streak for Isolation

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 Pseudomonasaeruginosathat you inoculated last week and streak for isolationanother 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?

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 oldMASTER PlatePP1 to grid square #1 of your newGram positivePP2, and repeat for all the colonies. Skip the numbers on 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.

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.

1. 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 mlE. 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 flasks1- 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 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.

1. 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.

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.