3 Lab 3: Enumeration and CFU/ml Calculation

Lab Objectives

After completing this lab, the student should be able to:

  • Use aseptic technique to prevent contamination during serial dilution.
  • Use aseptic technique to capture a “clean catch” urine specimen.
  • Explain serial dilution.
  • Apply the CFU/ml calculation (CFU/ml, CFU/g, PFU/ml, or PFU/g) to cell counts obtained from a serially diluted stock culture or specimen to determine the total number of viable organisms in the specimen.
  • Compare and contrast each of the following terms related to dilution and enumeration: isolation streak with serial dilution, total counts with viable counts.
  • Calculate CFU/ml (CFU/g, PFU/ml, or PFU/g) based on experiment plate counts.

Introduction

Enumeration techniques allow for microbiologists to count the number of viable cells within a sample.  Oftentimes samples will contain too many cells to count within the stock culture or original patient sample, thus requiring serial dilution before conducing cell counts.  Common enumeration methods include:

  • Standard Plate Count Method – which combines serial dilution with either spread plate technique or pour plate technique to serial dilute a sample and make it more countable.  This method allows for viable counts in that only living cells can divide and produce colonies.
  • Direct microscopic counts – use a microscope and a Petroff Houser counting chamber which is a special microscope slide that contains a grid.  A sample is loaded into the Petroff Houser counting chamber and a specific number of grids are counted which the total number of cells being counted in that number of grids and the average number of cells being used to calculate the cell density (number of cells) of the source specimen.  Direct counts give total, rather than viable, cell counts because it is difficult to determine if all cells are alive within a sample under phase contrast microscopy, especially if the sample contains nonmotile cells.
  • Spectrophotometer measurements – rely upon the use of a spectrophotometer to determine total cell counts within a sample.  For best results, spectrophotometric assays should be combined with viable plate counts (e.g., the standard plate count method).  A spectrophotometer works by detecting the amount of light that can pass through a sample – the more light that passes through the sample, the fewer cells are in that sample.  Likewise, a dense sample that contains many cells would block most of the light from passing through there by giving a low percent transmission (%T).

Viable vs. total cell counts are helpful in determine effectiveness of treatment and stage of disease progression.  Recall from lecture that pathogens would increase in cell number until the crisis point is reached.  The crisis point will decide if the patient’s immune system can overcome the infection, often with the use of chemotherapy (medication).  If the patient’s immune system cannot over the pathogen then pathogen numbers would continue to increase and the patient would not recover from illness.  Likewise, scientists can use viable counts to determine if a chemotherapy or physical control method can reduce the number of cells within a microbial population.  We’ll explore this theme a bit later on in the semester in the physical and chemical control labs.  In terms of treatment studies, viable counts are of greater value than total cell counts.

The standard plate count method can use either pour plates or spread plates.  The different lies in how the sample is inoculated into/ onto the agar and the amount of sample that can be inoculated.  Pour plates use tempered agar which is still liquid but cooled to around 45-50⁰C so as not to kill microbial cells when the sample is inoculated.  Pour plates typically have 1ml of inoculum added to the molten agar which is then mixed and quickly poured into empty sterile petri plates.  The plates are allowed to cool and solidify, which happens around 40⁰C before inverting and incubating.  Spread plates use traditional solid agar plates and have 0.1ml of inoculum added to the plate before the sample is spread or smeared across the agar surface using a cell spreading rod which is a bent rod made of autoclavable plastic, metal, or glass that is used to spread the inoculum across the surface of the plate.  Spread plates only take a few seconds to “dry” once the inoculum has been smeared before the plates can be inverted and incubated.  The amount of inoculum to be added to the plate often is the determining factor between spread plates and pour plates though other factors such as the amount of oxygen needed by cells within the sample should also be considered.  Because cells will be embedded with the pour plate methods, this method would not be idea for strict or obligate aerobes and the spread plate method should be used instead.

Dilution allows for samples that contain a high number of cells to be reduced to countable numbers.  Current CDC (2015) standards recommend that a plate count range of 25-250 cells per plate be used.  This means that plates that have over 250 colonies are reported as either TMTC (too many to count) or TNTC (too numerous to count) due to the likelihood of missing a colony in the counting process as the plate would be overcrowded.  Plates that contain less than 25 colonies would be reported as TFTC (too few to count) due to the statistical likelihood that a dilution error would inflate counts.

It should be noted that the isolation streak, discussed in a previous lab, also allows for sample dilution.  However, rather than depending on setting up a series of dilution tubes to achieve the dilution, the original source material (stock culture or specimen) is diluted as it is streaked across the surface of a plate.

While both plate counts and isolation streaks can be carried out on a variety of agar depending on the type of cell growth desired (refer to Lab: Aseptic Technique for a review of media types), specific general purpose agars such as PCA (plate count agar), TSA (tryptic soy agar), R2A (Reasoner’s 2A agar), or NA (nutrient agar) tend to be used for the standard plate count method.  These general purpose agars tend to have sufficient nutrients to grow most non-fastidious organisms within a sample.  Of course, enrichment agars would need to be used for patient samples from throat, sputum, or blood specimens, and selective agars would need to be used for fecal samples.

Now that the basics of what a serial dilution is, what it does, and how to make one have been reviewed, let’s discuss the CFU/ml calculation.  It should be noted that CFU/ml is used to determine the number of viable bacterial cells within a liquid sample while CFU/g would be used to determine the number of viable bacterial cells within a solid sample.  Likewise, bacteriophage counts can also be done from liquid cultures of bacteria (PFU/ml) and solid cultures of bacteria (PFU/g).

The equation for determining the counts/ volume (e.g., CFU/ml, CFU/g, PFU/ml, or PFU/g) is the same as are the counting limits (25-250 colonies for bacteria counts or plaques for phage counts) regardless of the original source material.

Equation 1: CFU/ml (CFU/g, PFU/ml, or PFU/g) Equation.

CFU/ml = average number of colony or plque counts(amount plated∗overall dilution)/average number of colony or plque counts(amount plated∗overall dilution)

It should be noted that plate counts are most commonly done in replicates – that is the same dilution would be plated onto more than one plate so that an average count per dilution could be obtained.  For the purposes of this lab, we will only be using one plate per dilution so the averaging of plate counts would not apply but you may need to average plates in CFU/ml problems on assessments.

Remember that the amount plated will vary depending on if spread plates (0.1ml plated) or pour plate (1.0ml plated) method is used.

The overall dilution would be the dilution of the sample plated compared to the stock culture.

Here is an example problem:

A urine specimen was obtained from a patient.  It was noted that the specimen contained traces of blood and appeared turbid indicating infection.  A culture and count were ordered.  The urine specimen was streaked for isolation on the MAC (MacConkey agar) using a four quadrant isolation streak to determine if fecal contamination was present.  One ml of the urine specimen was transferred to a 9ml dilution tube (1:10 or 10-1 or 0.1ml) was made and five subsequent dilutions were made in like manner.  From each dilution, two plates were inoculated with 0.1ml and the plates were grown overnight at 37⁰C before counting colonies.  Colony counts are reported in Table 1. This dilution can be illustrated as follows in Table 1.

Page Break

Table 1.  Serial Dilution of a Patient Urine Specimen. 

Tube 
Stock 
S1 
S2 
S3 
S4 
S5 
Amt. transferred to dilution blank 

0ml

1ml

1ml

1ml

1ml

1ml

1ml

Dilution 

0

10-1

10-1

10-1

10-1

10-1

10-1

Overall Dilution 

0

10-1

10-2

10-3

10-4

10-5

10-6

Amt. plated 

0.1ml

10-1

10-1

10-1

10-1

10-1

10-1

Avg. colony counts 

TMTC

TMTC

TMTC

218

27

TFTC

0

According to this data, plates from dilution S2 and S3 are within the countable range of 25-250 colonies so either of these counts could be used to determine the CFU/ml.  It doesn’t matter which dilution that is counted, they will be similar counts.  Because bacterial populations can be quite large and change rapidly, counts that share the same power of 10 are considered “equal” in the sense that the counts are close enough to convey needed information without causing statistical problems.  For example, a count of 1.37*105 and 7.8X*105 would both be useful in conveying that the cell density of the original urine specimen is 105. 

Let’s calculate the CFU/ml of this urine specimen using the S2 dilution where S2 has an average colony count of 218.

CFU/ml = 218/(10−1∗10−3)

CFU/ml =218/(10−4)

CFU/ml = 218 * 104 [Remember that the exponent will change signs due to division.]

CFU/ml = 2.18*106 [It is conventional to write the answer in scientific notation.]

What would be the CFU/ml if the S3 dilution were used instead? Use the space below to calculate the CFU/ml using S3 counts.

It is important to practice aseptic technique in both obtaining a urine specimen and in processing a urine specimen.  The clean catch technique is used to obtain a urine specimen for a non-catharized patient.  If a patient is catharized then aseptic technique would have been met at the time of catheter insertion.  For a urine specimen to be collected using the clean catch method, the area surrounding the urinary meatus (the area where urine leaves the body) should be cleaned with an antimicrobial or obstetrical wipe to remove any debris such as dead skin or microbial cells that could potentially contaminate and inflate bacterial counts.  Once the urinary meatus is cleaned, a small amount of urine should be voided into the toilet before collecting a urine specimen.  It should be noted that the entire urine specimen cup does not need to be filled – only enough to provide a sample for testing so aim for about 1/3 to 1/2 of the sample container.

Urine specimens can be used to determine quite a bit of information including, but certainly not limited to, leukocytes, urobilinogen, proteins, pH, blood, specific gravity, ketones, bilirubin, and sugars, all of which could indicate a diseased state.

In today’s lab, we will be testing our urine specimens to fecal contamination using a MAC plate, counting bacterial cells in our clean catch samples to determine if infection is present and if we have carried out the clean catch method correctly, and testing urine for leukocytes, nitrates, urobilinogen, protein, pH, blood, specific gravity, ketone, bilirubin, and glucose.  Students will work with their own samples to minimize health hazards.  Be sure to practice good aseptic technique throughout this lab.

Method (Lab@Home)

For this lab you will need:

Bathroom cups

Water

Dark beverage

Lab Methods

Paper

Cell phone

 

Instructions for serially diluting a specimen

You will need to disinfect your work space and wash your hands before completing this lab.  It is not necessary to work near a lit candle as you will not be handling cultures.

1. Using paper, make a label to sit in front of five different bathroom cups.  Your labels should include:

  • Stock – this will be the undiluted sample
  • D1 – this will be the first diluted sample
  • D2- this will be the second diluted sample
  • D3- this will be the third diluted sample
  • D4 – this will be the fourth diluted sample

2. For D1-D4 cups, add enough water so that the cups are half full.  You will use these “dilution blanks” in a moment.

3. Pour a volume of dark liquid into the bathroom cup until the cup is approximately half filled.  The choice of liquid is up to you but might include coffee, a dark colored soda, or juice.  The purpose of the dark liquid is to create contrast.

4. Pour half of the volume from the “Stock” cup into the “D1” cup.  You may want to gently swirl the D1 cup to mix the contents.

5. Pour half of the volume from the “D1” cup into the “D2” cup. Gently swirl the D2 cup to mix the contents.

6. Pour half of the volume of from the “D2” cup into the “D3” cup.  Gently swirl the D3 cup to mix the contents.

7. Pour half of the volume from the “D3” cup into the “D4” cup.  Gently swirl the D4 cup to mix the contents.

8.  Using the camera application on your phone (or other device), take a picture of your serial dilution.  You will want to submit this image along with the writing up of serial dilution to the assignment “Tech Exam: Serial Dilution”.

Work area clean up:

Once you have completed the activity,

  • Pour out the stock and dilution samples.
  • Throw away used cups.
  • Disinfect your bench.
  • Wash your hands.

Results

The results for this assignment will be uploaded into the assignment “Tech Exam: Serial Dilution”.

Table 3. Results of serial dilution of urine specimen.

Tube
Stock
I
S1
S2
S3
S4
S5
Amt. transferred to dilution blank

0ml

1ml

1ml

1ml

1ml

1ml

1ml

Dilution

0

10-1

10-1

10-1

10-1

10-1

10-1

Overall Dilution

0

10-1

10-2

10-3

10-4

10-5

10-6

Amt. plated

0.1ml

10-1

10-1

10-1

10-1

10-1

10-1

Colony counts

 

 

 

 

 

 

 

Use the space below to calculate the CFU/ml of your urine specimen.

Figure 2.  Results for UriTest urine test strips.  Source: https://healthywiser.com/products/0urinalysis-reagent-strips-urs-150ct

Circle your results for the UriTest strip in Figure 2 and record the purpose (meaning) for each of the urinalysis tests.  In other words, if the urine test is positive for leukocytes in the urine specimen, what does this indicate?  You may need to use the internet or other sources to answer this question.

Table 4. Results of UriTest strip.

Test
Meaning
Leukocytes

 

Nitrate

 

 

Urobilinogen

 

 

Protein

 

 

pH

 

 

Blood

 

 

Specific gravity

 

 

Ketone

 

 

Bilirubin

 

 

Glucose

 

References

CDC (Centers for Disease Control and Prevention). (2015). Infection Control: Appendix C. Water. https://www.cdc.gov/infectioncontrol/guidelines/environmental/appendix/water.html

License

Icon for the Creative Commons Attribution 4.0 International License

BI302 Clinical Microbiology Lab Manual by Dr. Lisa Ann Blankinship is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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