8.2: Food Microbiology

Learning Outcomes

By the end of this laboratory period, you should be able to

• Calculate the number of colony-forming units (CFU) in 1 gram of food using serial dilutions and plate counts.
• Explain how the data obtained from a standard plate count and a Hektoen or EMB plate are different and why they are both useful
• Describe situations where microbes in food are dangerous, and compare them to situations where the presence of microbes in food is deliberate.

Introduction

Every time you take a bite of food, microorganisms - sometimes millions of them - enter your body with the food. If the food is fermented, like yogurt or kimchi, the organisms were added on purpose to transform the food into a fermented product. However, even a bite or two of salad is usually accompanied by an abundance of microorganisms. This is not a problem…usually. Unfortunately, there are a few pathogenic microorganisms that utilize your gastrointestinal tract as a portal of entry. These organisms including Norovirus, Salmonella, E. coli O157:H7, and others, can wreak havoc on your body causing misery, serious illness, and occasionally death.

As a result of this, there are methods to determine how many microbes are in food, and what kinds of microbes are there. The “who” is usually more important than the “how many” since a few dangerous organisms are far riskier to health than an abundance of non-pathogenic bacteria.

Today, we will explore six different kinds of food, and ask the questions:

1. How many organisms (colony-forming units or “CFU”) are present per gram?
2. Do the organisms found in the food share metabolic features such as hydrogen sulfide formation with organisms like Salmonella? Are there an abundance of facultative anaerobes consistent with the kinds of organisms found in sewage?

The “How Many” Question - Serial Dilutions and Standard Plate Counts

Serial Dilutions

The first thing we are going to do is perform a serial dilution of the organisms in/on the food. This is accomplished by weighing out a specific amount of the food and diluting it in water several times to yield a range of concentrations. In our case, we will weigh out 1 gram of each type of food and dilute it three times, each by 100x. This gives us three different dilutions:

• 1/100 dilution (the original sample is diluted one hundred times)
• 1/10,000 dilution (the original sample diluted ten thousand times)
• 1/1,000,000 dilution (the original sample diluted one million times)

The next step is to determine how many organisms or “CFU” are present in 1 gram of each dilution. Not every organism on the food is viable or will be able to grow under the standard laboratory conditions that we use. So a “CFU” by definition means it’s an organism that is capable of forming a colony under our specific media and incubation conditions.

To determine how many CFU are present, we will take 1 mL or 0.1 mL of each dilution above and grow any viable organisms in TSA agar.

Figure \(\PageIndex{1}\): Schematic diagram of how to set up serial dilutions and plates to determine CFU/gram of each type of food

Referring to Figure \(\PageIndex{1}\) - you can see how 1 gram of food is diluted into 99 mL of water. Water weighs 1g/mL, so that’s the same as 99 grams of water or a 1/100 (1 g food/100 g total) dilution. If you take 1 mL of that dilution and prepare a TSA plate with that sample, the number of colonies (CFU) on that plate will be 1/100th the number of CFU per original gram of food. The same calculation can be made for the 10-4 and 10-6 dilutions. Although we are not preparing 1/1000 (10-3) or 1/100,000 (10-5) dilutions, we can easily prepare plates representing these dilutions by plating only 0.1 mL (one-tenth of a mL) of the 10-2 and 10-4 dilutions respectively.

After incubation for 24-48 hours, visible colonies will appear on the plates. The challenge now is to count them! Each plate should contain approximately 10x fewer colonies than the plate before. If your 10-2 plate contained 1000 colonies, you would expect your 10-3 plate to contain only 100 colonies, because you added 1/10th the amount of volume to that plate. This is true for each successive plate prepared in this experiment.

It’s impossible to count a plate with 1000 colonies. In fact, it’s not really possible to accurately count a plate with more than 300 colonies - we consider a plate with more than 300 colonies “too numerous to count” or “TNTC”. By the same token, a plate with fewer than 30 colonies does not have a statistically significant number of organisms present to provide a reliable count, and these plates are considered “too few to count” or TFTC. Therefore, in the 5 plates that we prepare, we are hoping that one or two of them contains between 30-300 colonies so that we can use them to calculate the number of CFU/gram in our original food sample.

Figure \(\PageIndex{2}\): Example data set. To calculate the number of CFU in the original sample, you would take 250 CFU and multiply it by ten thousand, or 35 and multiply it by a million. Together, this gives you a final answer of around 2-4 million CFU per gram of food.

The “Who” Question

In Lab 12, we learned about two types of differential media that are both commonly used in food microbiology and wastewater sampling - Hektoen and EMB. We will be using this media again today for a qualitative assessment of the kinds of organisms that are present in each type of food.

Eosin-Methylene Blue Agar

EMB (eosin-methylene blue) agar is frequently used to identify fecal coliform bacteria and is both selective and differential. It can be very useful in distinguishing a vigorous fermenter such as E. coli, from other fermenters. The dyes present in the medium including Eosin Y and methylene blue, inhibit the growth of Gram-positive bacteria. These dyes are also pH indicators that will form a dark purple precipitate at low pH. EMB plates also contain both sucrose and lactose. Organisms that ferment these sugars, and lower the pH, will turn purple on this medium. However, when the pH drops very low, below 4.9, the Eosin Y and methylene blue will form a complex with a green color. Very vigorous fermenters, such as E. coli, will form colonies with a green metallic sheen.

Figure \(\PageIndex{3}\): Typical growth on EMB. Left - a non-lactose fermentor. Right - a vigorous lactose fermenter such as E. coli produces a characteristic green sheen.

Hektoen Enteric Agar

Hektoen enteric agar is both selective and differential and was designed primarily to differentiate between Salmonella and Shigella in human specimens. It contains bile salts, Bromothymol blue, and acid fuchsin, all of which select against the growth of Gram-positive bacteria. It also contains Ferric ammonium citrate or thiosulfate, which will react with hydrogen sulfide (H₂S) to form a black precipitate - similar in principle to the reaction that occurred in the SIMs tubes in Lab #11.

Hektoen also contains lactose, sucrose, and salicin. Organisms such as E. coli will ferment these sugars producing acid, which will turn the Bromothymol blue an orange or salmon color. In theory, neither Salmonella nor Shigella will ferment these sugars, and in fact, they don’t in our phenol red broths. However, we often see Shigella turning orange on Hektoen in our lab, so don’t use an orange colony on Hektoen to rule out Shigella.

Figure \(\PageIndex{4}\): Left - Salmonella enterica produces hydrogen sulfide and turns black on Hektoen. Right - Shigella sonnei is not supposed to ferment lactose...but in our lab it always does producing acid and turning the medium orange.

Materials

Per Student Group:

• Food sample (ground beef, ground poultry, fresh spinach, fresh lettuce, yogurt, or kombucha).
• Plastic weigh boat
• Digital Scale
• Alcohol Wipes
• Forceps
• Pipette pump
• 3 x Glass or plastic mL Pipettes (Do not obtain until you are ready to use each one so that they
• remain sterile. Use plastic pipettes for ground beef, yogurt, and ground poultry.
• 3 99 mL sterile water blanks
• 5 sterile Petri dishes (empty)
• 5 molten TSA deeps equilibrated to 60^oC
• Cell spreaders
• EMB agar plate
• Hektoen agar plate

Experiment

Day 1

For steps 1-10, refer to Figure \(\PageIndex{1}\).

Prepare Dilution blanks

1. Collect three 99 mL water blanks and label them 10-2, 10-4, and 10-6.
2. Weigh out 1 gram of the food sample you’ve been assigned. Try to avoid adding any organisms to the food! Wipe out the weigh boat with an alcohol wipe, dip forceps into alcohol, and flame before using them to handle or tear food. Wear gloves! Do not touch the food with your bare hands.
3. Place the food (1g) into the 10-2 dilution blank. Mix thoroughly.
4. Using a 1 mL glass or plastic pipette, transfer 1 mL of the 10-2 dilution blank into the 10-4 dilution blank. Mix thoroughly.
5. Using a fresh 1 mL glass or plastic pipette, transfer 1 mL of the 10-4 dilution blank into the 10-6 dilution blank. Mix thoroughly.

Prepare Plates

1. Collect 5 empty Petri dishes, and label them with your names, dates, and the dilution they represent (10-2, 10-3, 10-4, 10-5, 10-6).
2. Ensure that each dilution blank is well-mixed. Using correct aseptic technique, pipette
   • 1.0 mL of the 10-2 dilution into the 10-2 Petri plate
   • 0.1 mL of the 10-2 dilution into the 10-3 Petri plate
   • 1.0 mL of the 10-4 dilution into the 10-4 Petri plate
   • 0.1 mL of the 10-4 dilution into the 10-5 Petri plate
   • 1.0 mL of the 10-6 dilution into the 10-6 Petri plate
3. Retrieve molten TSA deeps one at a time from the dry bath. Pour one deep into each plate, swirling gently (make a Figure \(\PageIndex{8}\) with the plate) to mix the dilutions with the agar. Do not swirl so hard that the agar touches the plate lid.
4. Allow the agar to cool and solidify on the lab bench.
5. Stack the five plates and secure them with a rubber band. Incubate at 37 ^oC for 48 hours or at 30 ^oC for 5 days.

6. Prepare Hektoen and EMB Plates

Pipette 0.1 mL of your 10-2 dilution onto one Hektoen and one EMB plate. Use the cell spreading technique to evenly distribute any bacteria across the plate. Stack the two plates and secure them with a rubber band. Incubate at 37 ^oC for 48 hours or at 30 ^oC for 5 days.

Day 2

Inspect your five dilution plates. Determine which ones have between 30 and 300 colonies, and count the number of colonies present on those plates. Complete the table below. Remember that if the plate contains more than 300 colonies, you will record “TNTC”. If there are fewer than 30, record “TFTC”.

Data

Calculate the number of CFU per gram in the original sample:

CFU per plate X dilution factor = CFU per 1 gram original food sample

Your data

Food Sample _______________________________

Table 1: Student Data From Standard Plate Counts

	10-2	10-3	10-4	10-5	10-6
CFU/plate
CFU per original 1 g food

EMB/Hektoen Plate Results:

Table 2: Student Data from Hektoen and EMB Plates

Bacteria Types	Plate type used for determination	Present	Absent
E. coli
Lactose Fermenters
Salmonella spp.

Class Data:

Record as CFU/gram of the original food sample

Table 3: Class Data from Standard Plate Counts

	Plates
Food Type	10-2	10-3	10-4	10-5	10-6

Questions

1. Based on the class data, which type of food had the highest CFU per gram? Does this make sense based on the type of food?
2. Based on the class data, which type of food had the lowest CFU per gram? Does this make sense based on the type of food?
3. Did any food contain fermenters? Would you expect this type of food to contain microorganisms that ferment sugars?
4. Did any food contain putative E. coli or Salmonella based on EMB and Hektoen data?
5. Do you consider any of these foods to be unsafe to eat? Explain your answer.