Introduction to culturing

The process of culturing bacterial samples and analyzing colonies

An overview of pH value and buffer solutions

Before the culturing process can begin, bacteria must be diluted serially with a buffer solution. The process begins with collected samples of bacteria being added to a buffer solution. A buffer solution helps maintain the 7 pH. An example would be phosphate buffer saline. Phosphate buffer saline (PBS) is an alkaline solution, meaning it is basic. Ratzke C, Gore J (2018). A buffer solution consists of a weak acid (HA) and its salt conjugate base, or a weak base (A) and its salt conjugate acid. It is made from a weak base and a salt. It helps maintain 7.2-7.4 pH for biological samples. On a pH scale, 0 pH is acidic, 14 pH is basic and 7 pH is neutral. 7.2 pH is slightly on the basic side. Phosphate buffered saline is a buffer commonly used in biological research. It is a water-based salt solution composed of sodium chloride (NaCl), sodium phosphate (Na3PO4), and (in some formulations) potassium chloride (KCl) and potassium phosphate (K3PO4). Aryal, Sagar (2018). It is an isotonic solution, meaning that it is in equilibrium, so no changes to the cells will occur (compare to hypertonic and hypotonic). Agar plates are common media used in the lab for growing microorganisms such as bacteria. Agar is commonly used for biological samples because it can grow a wide variety of bacteria. Agar is combined with nutrients that aids in growth and are poured onto plates and solidifies. For the culture, the solution from the test tubes is added to the surface of the agar plates. Aryal, S., Emmanuel, O., Anusree, Onuchi, O. A., David, Bradbury, S.,Jk (2018). Gently sweep a sterile inoculating loop across the surface in a rigid sinusoidal pattern. Try to distribute the sample proportionally. For a quadrant streak, which may be necessary in some cases, divide the Petri dish into thirds or fourths to gradually reduce the number of microorganisms that can occupy that region, producing single colonies instead of an unquantifiable bacterial lawn. Spread plating does not additionally dilute samples; a sterile glass spreader is used to distribute suspension (test solution) media across an entire petri dish. The colonies that grow on the spread plate arise from a single cell and each colony on the dish can be counted to estimate the number of colony-forming units in a given suspension (CFU/mL). Nutrient agar is the surface media in which PBS and the bacterial samples will be added to in order to promote bacterial growth.

Nutrient agar ingredients

0.5% Peptone: Peptone is the principal source of organic nitrogen for the growing bacteria.

0.3% beef extract/yeast extract: Water-soluble substances that aid in bacterial growth, such as vitamins, carbohydrates, organic nitrogen compounds, and salts.

1.5% agar: The solidifying agent.

0.5% NaCl: It maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms.

Distilled water: Water is essential for the growth and reproduction of micro-organisms. Aryal,S (2018).

How to prepare nutrient agar

1. Suspend 28 g of nutrient agar powder in 1 liter of distilled water.

2. Heat this mixture while stirring to fully dissolve all components.

3. Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.

4. Once the nutrient agar has been autoclaved, allow it to cool but not solidify.

5. Pour nutrient agar into each plate and leave plates on the sterile surface until the agar has solidified.

6. Replace the lid of each Petri dish and store the plates in a refrigerator.

pH is adjusted to neutral (7.4) at 25 °C.

Serial dilutions and plating

As explained in our procedure, serial dilutions are purposed for dilution of the number of bacteria/volume of buffer. In this case, in order to analyze the colony-forming units (CFU's) on an agar plate, the solution needs to be diluted enough so that there will be between 30-300 CFU's on the plate. An example would be if you have a test tube rack containing six test tubes and the first test-tube contains the original sample of bacteria with 10mL of buffer solution. The other five test tubes will have 9mL of buffer solution, but no bacteria yet present. 1mL of solution will be pipetted from test tube 1 and placed into test tube 2. Then 1 mL of solution is taken from test tube 2 and placed into test tube 3. This dilution series decreases the ratio of bacteria per volume of the buffer by a 10 fold. The 5th and/or 6th test tube will be diluted enough that when 1mL of solution is taken from each and spread onto separate agar plates. Colonies of bacteria are then spread onto the surface of the agar in a sinusoidal pattern, traced over about 3-4 times in quadrants with an inoculation loop. Karcher, S. J. (1995). Store Petri dishes in a refrigerator upside down and cover with. After an incubation period of 24 hours, colonies should appear. Cover with parafilm over the surface. This keeps condensation from dropping onto the bacteria. Let dishes come to room temperature before use. Let grow in an undisturbed location. Bacteria can grow at any temperature from about ambient room temperature (hopefully around 70°F) all the way up to about 100°F. Do not place in sunlight or on a heater. The dishes will start to smell when the bacteria are growing. Make observations and keep records of what you see growing in each dish. Colonies on an agar plate are referred to as colony-forming units, or CFU's. A colony is a visible mass of microorganisms all originating from a single mother cell. A colony is made up of clones of bacteria all genetically alike. Bacteria are prokaryotic organisms, therefore single-celled. They reproduce by binary fission. This is why the colonies are clones; binary fission is when the DNA of the bacterium replicates by dividing in half. The bacterial cell elongates and splits into two daughter cells, each with identical DNA to the parent cell. The number of CFU's on the plate being analyzed must be between 30-300 CUF's. This is the ideal range in order to have accurate data and to quantify. Once bacterial colonies have grown, the analysis collection can begin.

Equations for counting bacteria

Individual Serial Dilution (INSD): Calculate the number of CFU's on the countable plate, the number of microorganisms per mL. (INSD)=Number of colonies/dilution factor

Total Series Dilution Factor (TSDF): Calculate how much the original sample was diluted total (TNSD). *Do NOT include any dilutions after the countable plate. The total series dilution factor is a calculation of how much the sample was diluted in all of the tubes combined. This is accomplished by multiplying each of the appropriate INSD. This series does not include any dilutions after the countable plate. (TNSD=INSDx)

Plated dilution factor (PDF): calculate the dilution factor once the sample is plated (CFU/mL). (PDF = mL plated/1 mL)

Final dilution factor (FDF): how much the original sample was concentrated. (FDF = SDF x TSDF x PDF)

Colony-forming units (CFU/mL): To find how many bacteria were in the original sample. (CFU/mL = (CFU's on the countable plate) x (1/FDF))

Analyzing bacterial cultures

Make sure to keep the petri dish covered with parafilm and record observations in a notebook. Acquiring photographs of the colonies may be helpful as well. Colonies are distinguishable according to their shape, size, color, and texture. . Look for differences and similarities. The colonies observed may look different, showing different types of bacteria. Color, shape, texture are some examples of what to look for when observing.

What to analyze

Form – the basic shape of the colony (circular, filamentous, etc.)

Size – the diameter of the colony. Tiny colonies are referred to as punctiform.

Elevation – this describes the side view of a colony. Turn the Petri dish in the end.

Margin/border – the edge of a colony. What is the magnified shape of the edge of the colony?

Surface – how the surface of the colony appears (smooth, glistening, rough, wrinkled, or dull.)

Opacity – transparent (clear), opaque, translucent (like looking through frosted glass), etc.

Color – white, buff, red, purple, etc.



Bacterial colonies on the same agar plate may stay separated, or some may merge. The example of B. subtilis 168 shows a single strain colony. This means it is made up of one single bacterial cell in which replicated due to binary fission while growing on the nutrient agar. The neighboring colonies show two separate strains of bacteria. The colonies look different, therefore they are not the same type of bacteria (Leung, B., & Liu, S).