#### Estimated time usage: 60 min/45 min
## Background
The easiest and most common way to evaluate the microbial attributes of a product is to count numbers of different bacteria or fungi in the product. Since microbes are small and typically unevenly distributed in products, a couple of common steps are used to analyze their microbial attributes. The first step is to obtain representative food samples and homogenize the samples with buffer. The homogenization step is designed to release the microbes from the food samples (think about milk vs. condensed milk vs. cheese, **Figure 1.1A**). After homogenization, sample suspensions can be further diluted for plating onto agar plates. Making dilutions will allow one to have countable plates for the final calculation (**Figure 1.1B**).
Figure 1.1 (A) Pictures of milk and cheese samples; (B) plates with countable colonies vs. plates covered with bacterial lawn. Photos in (A) were obtained via Unsplash.com.

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### Plating methods
There are different plating methods, including streak plating, pour-plating, and spread-plating. Streak plating, also called streak for isolation, is used to isolate single colonies of microbes (**Figure 1.2A**). Spread-plating and pour-plating are for obtaining microbial counts of the samples (**Figure 1.2B**). While spread-plating and pour-plating can be used interchangeably in most cases, the pour-plating method has the following advantages: 1) It allows the growth and quantification of microaerophiles, as there is less oxygen in the agar plate than on the agar surface; and 2) more surface area is provided as the sample is mixed and spread throughout the media. For spread-plating, 100 µl of sample is used. For pour-plating, 1 ml of samples is added.
<center>Figure 1.2 (A) Scheme of streak plating. Created with BioRender.com</center>

<center>Figure 1.2 (B) Scheme of spread and pour plating: top, steps to follow for spread-plating; bottom, steps to follow for pour-plating. Created with BioRender.com.</center>

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There is no one medium or mode of incubation that will support the growth of all bacteria. The final microbial numbers you obtain for one product are dependent on the media you use and the incubation conditions (e.g. temperatures and time). In addition, the presence of antagonistic and/or mutualism species in the samples, the proportion of injured cells, the food samples you analyze (as discussed earlier, solid vs. liquid samples), as well as the tendency of bacteria to clump all impact your final counts. The tendency of bacteria to clump means the phenomenon of cells depositing on the plate as a group and forming a single colony indistinguishable from those formed from individual cells.

As mentioned in the previous paragraph, the selection of media (agar plates) for plating and growing bacteria can directly impact the final bacterial counts you obtain. Media selection will also determine the types of bacteria that grow on the media. In general, all microorganisms require a source of metabolic energy, a source of carbon and nitrogen to build cell materials, and numerous minerals. Some bacteria can grow on a simple medium containing a single organic compound serving as both the energy and carbon source, with all other ingredients being inorganic. Most bacterial pathogens and parasites, however, do not fall into this category. Since these bacteria rely on their host for certain organic compounds rather than synthesizing their own, a more complex medium is used when growing them in the laboratory. In the FST104L manual, you will see that I have included the basic recipe for the media used in each lab. The purpose of doing so is to familiarize you with the ingredients used in these media. Since complex media (generally called non-selective, non-differential, or nutrient media) can support the growth of a large number of microorganisms, selective ingredients such as bile salts, sodium deoxycholate, and sodium lauryl sulfate are added. The three chemicals listed here are used to inhibit the growth of Gram-positive bacteria. You will notice that different selective agars will have different chemicals added for the selection of target bacteria, thus they are called selective media. Sometimes, chemicals may be added simply for changing the pH. For example, potato dextrose agar (PDA) is used for growing molds and yeasts. If you don’t adjust the pH of ~5.6, bacteria can also grow on PDA. You may overestimate your fungal counts.

When conducting microbiological experiments, aseptic techniques are crucial to prevent the contamination of samples and ensure correct results from the analysis. It is also essential to protect the safety of laboratory personnel. Examples of aseptic techniques include cleaning and sanitizing your bench top every time before you start your experiment, wearing gloves when handling products, flaming the transferring metal loops between samples, minimizing the time period your samples and dilutions are exposed to air, and using new pipette tips for different dilutions (even for the same sample).

Finally, we need to talk about the presentation of the final counts. You will find detailed information about how to calculate total bacterial counts in your sample on pages 7-8 of this manual. Instead, I want to emphasize the presentation of the data. When presenting the final microbial counts of your samples, you always present the base ten logarithm of the counts. Make sure you use the correct unit. For example, if your final count is 100 bacterial cells in 10 ml of milk sample (100 CFU in 10 ml), then you present the data as 1 Log10 CFU/ml or 1 Log CFU/ml. Do not present 10 cells/ml.

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### Objectives
1. Practice proper aseptic techniques
2. Be familiar with general guidelines for labeling plates and tubes
3. Understand what to read on a media bottle (e.g. labels, instructions, directions, expiration dates, receiving and opening dates, etc.)
4. Plate food samples (e.g. cheese) to obtain total aerobic plate counts using pour-plating and spread-plating methods
5. Review and practice Gram staining techniques

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### Materials

**Food samples**: cheese
**Bacterial strains**: one Gram+ strain (*Bacilius cereus*) and one Gram- strain (*Escherichia coli*)
**Media**: Plate count agar (PCA) Supplies: **5** tubes of buffered peptone water (for making dilutions) **14** pre-poured plates for spread plating **14** empty plates for pour-plating Pipette and tips

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### Procedures [VP for lab 1](https://drive.google.com/file/d/1cabH53NUgBohDMGqJmC5FQ4LBCFoHrmw/view?usp=sharing)
**STEP 1**: Get yourself familiar with the typical tools and settings in a microbiology lab
- Clean and sanitize your bench top by spraying with 70% ethanol and wiping up.
- Orient yourself and get familiar with the safety features in the lab and in the building.
- Identify a place to store your bags.
- The supplies and tools are arranged for groups. Please check with the TA and identify the sets that belong to your group.

**STEP 2**: Aseptic technique review and practice
- Flame your transfer loops using Bunsen burners🔥 . **IMPORTANT Keep ethanol spray (any flammable reagents) far away from the Bunsen burners**.
- When working with capped bottles or tubes, remove the cap by manipulating between the small finger and the palm of your hand 👌🏻.
- Once the bottle or tube is open, always hold the cap in your hand rather than leaving it on the table. If you have to leave it on the table, leave it upside down so that the interior of the cap will not touch or face the table.
- When uncapping a microcentrifuge tube, use your thumb to pop open the lid. Avoid touching the inside of the tube.
- When pipetting liquid from the tube, do not dip the entire tip into the tube. Always keep your pipette upright so that the liquid will not enter the pipette.
- When streaking a plate, you aim to obtain an isolated colony. **The assumption is that each colony is developed from a single cell.** In this case, you have to flame your loop well to prevent contamination between two uses. Cool your loop before you dip it in the sample. Take you loop out and steak on to the plate as indicated in Figure 1.2A.

**STEP 3**: Plating your food samples
1. Weigh 10 grams of cheese and blend it with 90 ml of buffered peptone water (provided in bottles) with a sterile blender for 2 min. Dilute your samples with 4.5 ml of peptone water.
2. Label your plates with sample names and dilution factors (e.g. O: Original; 1/10 (10-1) to 1/100,000 represent dilution factors).
3. For the spread-plating method, get two pre-made PCA plates for each dilution. From each dilution tube, pipette two 100 µl of samples out and add them to the top of the two plates. Use a spreader and spread the liquid sample evenly on the agar surface.
4. For the pour-plating method, get two empty plates for each dilution. From each dilution tube, pipette two 1 ml samples out and add them to the empty plates. Add ~20 ml of cooled liquid PCA agar and move the plates gently around in a figure 8 pattern on the bench top to mix the sample and the liquid agar. ~20 ml is about the amount of agar that just covers the bottom of the plate. Let the agar solidify for at least 10 min.
5. Control plates. Control plates are plates that plated with sterile water. The goal of having control plates is to make sure no contamination has occurred during the operation. Label one pre-made PCA agar with “Control-S,” add 100 µl of sterile water onto the surface, and spread the sample evenly. Next, add 1 ml of sterile water to an empty plate, add ~20 ml of liquid PCA, mix the sample well, and let the agar solidify (label this plate “Control-P”).
6. Invert all plates and incubate at 30 ˚C for 48 hours. Count the colonies when you come to the next lab.

**STEP 4**: Streak plating
1. Streak plates for preparing your cultures for Gram staining on the next lab day. Take a loop and obtain a loopful of liquid culture from one tube. Each group only needs to obtain one tube (either *Bacilius cereus* or *Escherichia coli*). Streak the culture onto your plates as shown in Figure 1.2A. Invert the plate and incubate it at 30 ˚C for 48 hours. <center>**End of the first section of week** </center>

**STEP 1**: Colony counting and calculation
1. Using the guidelines [here](https://hackmd.io/@g4P5SSbiSriJQ-hJMcd9fg/rk9-pTNun), count the colonies on all plates. For plates with more than 250 colonies, use the colony counter and hand tally. The calculations outlined in the guidelines are based on the dimensions of the plastic petri dishes used in class, which have an area of about 56 cm2.

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Microscope and Gram Stain specific protocol can be found [here](https://drive.google.com/file/d/15tCPMxekoiHlgNO0d-eewjRBHdavL_NM/view?usp=sharing).
**STEP 2**: Gram staining
1. Add a drop of sterile water on a glass slide
2. Pick up a well isolated colony from your plate with a loop and mix it with your drop of water.
3. Air dry and heat fix it.
4. Add a couple of drops of Crystal Violet and keep it on for 1 min.
5. Rinse the Crystal Violet off with water and add a couple of drops of iodine. Leave the iodine on for 1 min.
6. Rinse the iodine and add a couple of drops of 95% alcohol or acetone. Leave it on for 20 seconds.
7. Rinse off the alcohol and add a couple of drops of Safranin. Leave it on for 1 min.
8. Rinse the Safranin off and blot dry your glass slide.
9. Observe your result with a microscope. <center>**End of the second section of week** </center>