LAB 26 AND 27(Data Sheet 4.5, 6.1, and handout)

STANDARD PLATE COUNT (p. 217 lab manual, exercise 6.1)

How do we count bacteria? There are many methods. Machines like a spectrophotometer can measure turbidity and thereby estimate the amount of bacteria in a sample. Coulter Cell Counters can count individual cells as they pass by a light beam. Standard Plate Count is a manual method, also known as a population count.

Practical Applications

  1. Medicine
  2. Diagnosing infections: a normal urine sample is expected to have 0-300 bacterial cells per ml. If the patient has more than 1000 cells it indicated infection. Bacteremia is the term for bacteria in the blood that are not multiplying. Septicemia is the term for bacteria in the blood that are multiplying. To determine the difference, we would need to do plate counts from a specimen drawn on a later date.
  3. Food Industry
  4. Quality Control: All foods have some bacteria. The County Health Department has decided what is an acceptable amount of bacterial cells per sample. A food (milk, hamburger, etc) is labeled “Grade A” when it has less fewer than the allowed amount of bacteria. The County does random, unannounced testing on samples.

Plate Counts are not qualitative because they do not tell you what organism is present. However, it is quantitative because it gives you the number of organisms present.

Counting always involves math.

Try to guess how many bacterial colonies are present in a flask that is turbid from E. coli.

Write your guess down in Standard Scientific Notation.

EXPRESSING NUMBERS IN STANDARD SCIENTIFIC NOTATION

For practice: How many zero’s are there? That is the number for your exponent.

1 = 100 = 1.0 x 100

10 = 101 = 1.0 x 101

100 = 102 = 1.0 x 102

1000 = 103 = 1.0 x 103

10,000 = 104 = 1.0 x 104

Now that you’ve had practice, let’s use a random number.

Just count how many digits there are after the first one to get your exponent.

Then round off the given number to the nearest 10th (2 digits):

10634 = 1.1 x 104

Negative exponents (10-1) are fractions less than one.

Positive exponents (101) are whole numbers greater than one.

Now let’s practice using negative exponents:

Round off the third number you come to (other than zero).

For instance, if the number is 0.00342, round down the number to 0.0034

If the number is 0.00345, round up the number to 0.0035

How many digits are there to the right of the decimal point before you get to a number greater than zero? That is your exponent number.

0.01= 10-2 = 1.0 x 10-2

0.00126 = 1.3 x 10-3

0.000542 = 5.4 x 10-4

PLATE COUNT PROCEDURE

Remove a set volume of the sample with a pipette. A pipette is an instrument that measures and delivers an accurate volume. Place the set volume sample in an empty Petri dish, add molten nutrient agar, and mix. The E. coli have flagella and are motile, so they will spread out a bit; however, they still tend to aggregate in clusters a bit.

Each group of cells (aggregate) forms a colony. These colonies are called Colony Forming Units (CFU). A CFU is a cluster (aggregate) of cells which eventually leads to the formation of a single colony. However, one single cell could also form a colony by itself. Therefore, by doing a Plate Count, we are unable to determine how many cells are present; we only count the colonies.

Suppose we transfer 1 ml of the E. coli sample to the Petri dish, and suppose that the correct amount of bacteria present in the solution is 2.5 x 109. That would mean there would be 2.5 billion colonies on that plate. That would be impossible to count!

COUNTABLE PLATE: 30-300 colonies

TMTC (Too many to count): 300+ colonies

TFTC (Too few to count): less than 30 colonies

To get our sample down to the number of colonies that we can count, we have to take less than 1 ml of sample. The pipettes we have can deliver 1 μl of sample, which is 1/1000 ml.

That would give us 2.5 million CFUs on the plate; still too many. Therefore, when the sample is too concentrated, we need to do a dilution series. But how much to we dilute? 250 CFU is a countable number.

DILUTION SERIES

This method takes a volume of a sample and dilutes it with a volume of a sterile liquid.

The sterile liquid is called “diluent”. It can be made from sterile water, saline, buffer, etc.

To calculate dilution, use this formula: D = V1

V2

D = Dilution factor

V1 = volume of sample being diluted

V2 = TOTAL volume after adding the diluent.

Look at page 219 of your lab manual and calculate the first dilution shown in the picture. Determine the dilution factor.

V1 = 0.1 ml

V2 = 9.9 ml of diluent + 0.1 ml of sample

D = 0.1  1/100  10-2

10

Therefore, D = 10-2

Suppose you take 10 μl out of the original sample and add it to 990 μl in a second sample.

V1 = 10

V2 = 990 + 10 = 1000

D = 10/1000 = 1/100 = 10-2

Therefore, D = 10-2

Note that you come up with the same dilution factor even if you use μl instead of ml.

Look at the plate values in the manual. 10-8 requires a dilution of a dilution of a dilution.

Calculate the dilution factors of the other dilutions in the exercise.

DILUTION SERIES

A dilution is to make something less concentrated by adding a liquid that contains none of what’s being diluted. The liquid is called the diluent. It causes the solution to become a fraction.

Sample Problem

If you start with 2 ml of E. coli and add 2 ml of sterile water, what’s the dilution?

D = 2ml / 4ml

D = ½

D = 0.5

D = 5.0 x 10-1

Remember that drinking alcohol is toxic! But if you drink, make sure you eat first. Then only drink one ounce of alcohol per hour. One ounce of liquor = 40% alcohol = 80 proof.

One beer = one ounce liquor = one glass (4 oz) wine.

Sample Problem

Let’s examine a very dangerous drink called Long Island Iced Tea and find out why this is so dangerous.

  1. Calculate the final % alcohol in Long Island Iced Tea
  2. How many hours do you have to sip it so that you don’t drink more than 1oz/hour?

Here is the recipe:

1 oz rum

1 oz gin

1 oz tequila

1 oz vodka

1 oz triple sec

1.5 oz sugar water with lime (sweet and sour mix)

0.5 oz Coca Cola

Solution

The total volume of alcohol = 5 oz

The total volume of the drink = 7 oz

The alcohol is 40% because it says on the bottles that they are 80 proof.

Therefore:

D = 5/7 = 0.71

0.71 x 40 = 28.4%

D = 28.4%

How many hours should you take to drink it?

5 (oz liquor) minus 0.71 (dilution factor) = about 4 hours 20 minutes

DILUTION SERIES

The goal is to determine the original cell density (OCD) in CFU/ml.

Cell density is actually cells per ml, but we can’t express it that way because more than one cell can form one colony, and we only count colonies.

Countable plate = 30-300.

Small samples are not an accurate representation of the true amount, so the data is discarded.

LOOK AT THE DIAGRAM ON P. 219 OF YOUR LAB MANUAL:

We will add 10 μl of E. coli to 990 μl of sterile water.

First, calculate the dilutions in each part of the series:

  1. D = 10/1000 = 1/100 = 10-2
  2. D = 10-2 x 10-2 = 10-4
  3. D = 10-4 x 10-1 = 10-5
  4. D = 10-5 x 10-1 = 10-6
  5. D = 10-6 x 10-1 = 10-7

Next, the Plating Factor is in ml, so we have to convert μl to ml.

1000 μl = 1ml

Therefore, divide each answer by 1000

A = 0.1 ml = 10-1

B = 0.1 ml = 10-1

C = 0.1 ml = 10-1

D = 0.1 ml = 10-1

Now apply the formula:

OCD (cfu/ml) = colony count

PF x FDF

Example: If the colony count for plate D is 56, what is the OCD?

Answer:

56___ = 56 = 56 x 108 = 5.6 x 109

10-1 x 10-7 10-8

Supplies needed for dilution series

990 μl pipette

900 μl pipette

100 μl pipette

10 μl pipette

5 sterile test tubes labeled 1-5

Sterile water

4 Petri dishes per lab group

Sterile molten agar

Bio waste bag

How to use the pipette:

The pipette has a look which hangs on your index finger.

It has a plunger on top which has two positions:

  1. Depress the plunger until you feel resistance and then stop
  2. Depress the plunger all the way.

It has a trigger lever on the side that discards the sterile tip.

PROCEDURE

Preparation of water samples

  1. Take the 990μl pipette and attach the sterile tip.
  2. Flame the neck of the sterile water container.
  3. Depress the plunger to position one (until you feel resistance).
  4. Insert pipette into the sterile water and suck up the sample, wipe excess off inside.
  5. Flame the mouth of tube 1
  6. Insert the pipette into tube 1 and depress all the way, wipe off excess.
  7. Flame the mouth of the tube and replace the cap.
  8. Discard the tip (use the trigger) into the biowaste bag
  9. Attach a new tip.
  10. Repeat for tube 2
  11. Take the 900μl pipette and attach the sterile tip.
  12. Flame the neck of the sterile water container.
  13. Depress the plunger to position one (until you feel resistance).
  14. Insert pipette into the sterile water and suck up the sample, wipe excess off inside.
  15. Flame the mouth of tube 3
  16. Insert the pipette into tube 3 and depress all the way, wipe off excess.
  17. Flame the mouth of the tube and replace the cap.
  18. Discard the tip (use the trigger) into the bio waste bag
  19. Attach a new tip.
  20. Repeat for tube 4
  21. Repeat for tube 5

Preparation of E. coli sample

  1. Take the 10μl pipette and attach the sterile tip.
  2. Mix the E. coli sample
  3. Flame the neck of the E. coli sample.
  4. Depress the plunger to position one (until you feel resistance).
  5. Insert pipette into the E. coli and suck up the sample, wipe excess off inside.
  6. Flame the mouth again, return stopper, and set the E. coli sample aside.
  7. Flame the mouth of tube 1
  8. Insert the pipette into tube 1 and depress all the way, wipe off excess.
  9. Flame the mouth of the tube and replace the cap.
  10. Discard the tip (use the trigger) into the bio waste bag
  11. Attach a new tip.

NOTE: MIX EACH OF THE TUBES WELL AFTER INOCULATION.

Dilution Series

  1. Use the 10μl pipette with the new sterile tip.
  2. Mix tube 1, remove the cap, and flame the neck.
  3. Depress the plunger to position one (until you feel resistance).
  4. Insert pipette into tube 1, suck up the sample, wipe excess off inside.
  5. Flame the mouth again, return stopper.
  6. Flame the mouth of tube 2
  7. Insert the pipette into tube 2 and depress all the way, wipe off excess.
  8. Flame the mouth of the tube and replace the cap.
  9. Discard the tip (use the trigger) into the bio waste bag
  10. Attach a new tip.
  11. Use the 100μl pipette with the new sterile tip.
  12. Mix tube 2, remove the cap, and flame the neck.
  13. Depress the plunger to position one (until you feel resistance).
  14. Insert pipette into tube 2, suck up the sample, wipe excess off inside.
  15. Flame the mouth again, return stopper.
  16. Flame the mouth of tube 3
  17. Insert the pipette into tube 3 and depress all the way, wipe off excess.
  18. Flame the mouth of the tube and replace the cap.
  19. Discard the tip (use the trigger) into the bio waste bag.
  20. Attach a new tip.
  21. Repeat with this pipette (with new sterile tip) for tubes 3  4
  22. Repeat with this pipette (with new sterile tip) for tubes 4  5

Plating Out the Sample

Label four Petri dishes A, B, C, D on the top and bottom.

Give your group a name and write this on the dish top and bottom.

Line your plates up on the demo table near the edge.

Deliver 100μl to each plate, starting with the lowest concentration so you won’t have to change tips. Low concentrations will not contaminate higher concentrations, but higher concentrations will contaminate lower concentrations.

  1. Take 100μl from tube 5 and deliver it to plate D.
  2. Take 100μl from tube 4 and deliver it to plate C
  3. Take 100μl from tube 3 and deliver it to plate B
  4. Take 100μl from tube 2 and deliver it to plate A

Get the molten agar (50°C) from the hot water bath (one bottle for 4 plates).

Flame the agar bottle neck and pour into dishes A, B, C, D, just enough so the bottom is covered.

GENTLY move the plate in a figure “8” six times. Make sure you don’t get the agar on the top of the plate.

Let sit until agar becomes opaque, and then set in crisper upside down.