13.16: Basics of Bacterial Culture

Bacterial Cultures

Bacterial culture is a fundamental technique in microbiology that allows the growth and study of microbial species under controlled laboratory conditions. This page outlines the physical and chemical conditions required for bacterial growth, media preparation methods, and standard protocols for handling and isolating bacteria.

Culture Conditions

Optimal bacterial growth depends on various environmental parameters, including temperature, pH, oxygen availability, and pressure. These conditions vary among bacterial species and must be controlled to ensure successful cultivation.

• Culture Temperature: varies depending on the organism’s optimal growth range
  • bacteria can be classified, based on temperature preferences, into several categories:
    • Psychrophiles grow best at 0–20°C
    • Mesophiles prefer 20–45°C (most human pathogens thrive at 37°C)
    • Thermophiles grow optimally at 45–80°C
    • Hyperthermophiles can tolerate temperatures >80°C
• Oxygen Levels: can vary among bacteria, depending on their metabolic needs and their ability to tolerate or neutralize reactive oxygen species
  • bacteria can be classified, based on their oxygen preferences, into several categories:
    • Obligate aerobes require oxygen
    • Obligate anaerobes are inhibited or killed by oxygen
    • Facultative anaerobes grow with or without oxygen
    • Microaerophiles require low oxygen concentrations
    • Aerotolerant anaerobes do not use oxygen but tolerate its presence
• pH: optimal pH depends on bacterial species
  • most bacteria prefer a neutral pH (6.5–7.5)
  • Acidophiles thrive in acidic conditions (pH < 5.5)
  • Alkaliphiles grow best in basic conditions (pH > 8.5)
• Pressure:
  • Barotolerant organisms can withstand increased pressure
  • Barophiles (also called piezophiles) require high-pressure environments (e.g., deep-sea organisms)

Culture Media

The culture media and reagents used in cell culture are designed to provide chemical support of the cells under sterile conditions. More information can be found in Chapter 7.2 Microbial Identification. Commonly used media and reagents include:

• liquid medium:
  • broth
• solid medium, including:
  • slant
  • deep (also called a stab)
  • plates

Liquid Medium (Broth)

Most bacteria can be grown in a basic nutrient broth under standard laboratory conditions. Nutrient broth is an example of a non-selective medium that provides essential nutrients to a wide range of bacteria. However, nutrient broth can be modified based on the specific requirements of the experiment. For example, antibiotics can be added to this medium to turn it into a selective medium. To read more about non-selective and selective media, go to Chapter 7.2: Microbial Identification. A basic nutrient broth can be prepared from individual components (see below) or from commercially available nutrient broth powders that simply require the addition of distilled water to reconstitute.

The components of a basic nutrient broth are:

• Peptone: source of organic nitrogen and growth factors
  • prepared from partially digested protein (typically from casein, meat, or soy)
  • used to cultivate a wide range of bacteria that do not require special nutrients
  • contains short peptides, amino acids, and some carbohydrates
  • can also buffer the liquid medium against pH changes by neutralizing acids made during microbial growth
• Beef extract: source of vitamins, minerals, and growth factors
  • complements the nutrients provided by peptone
  • contains water-soluble vitamins, especially B-complex vitamins, required for bacterial metabolism and enzyme function
  • contains phosphates, potassium, magnesium, iron, and sulfur compounds vital for enzyme function
  • contains a small amount of carbohydrates, organic acids, and lipids
• NaCl: maintains osmotic balance during bacterial growth
  • also stabilizes cell membranes and supports the function of membrane transport systems

Nutrient Broth Recipe

• to 900 mL of distilled water, add the following to a 2 L glass flask:
  • 5.0 g Peptone
  • 3.0 g Beef extract
  • 5.0 g NaCl
• place a magnetic stir bar in the flask and swirl gently on a stir plate until the components are fully dissolved
• adjust the pH to 7.0 ± 0.2 (if specified)
• adjust the volume to 1.0 L using distilled water
• autoclave at 121°C for 15 minutes
• store at 4°C
• if adding antibiotics, cool to room temperature (or 4°C) before adding.

Another equally popular non-selective liquid medium used in the growth of a wide range of bacteria are Luria-Bertani (LB) broth. LB broth is especially popular when growing E.coli bacteria that have been transformed with DNA. The recipe given below is sometimes called LB Miller broth. Modifications of this broth include LB Lennox broth (5.0 g NaCl) and LB Low Salt broth (up to 5.0 g NaCl).

The components of LB broth are:

• Tryptone: source of nitrogen and carbon compounds
  • partially digested casein that is produced using the enzyme, trypsin
  • provides short peptides and free amino acids
• Yeast extract: source of vitamins, growth factors, and minerals
  • contains essential B-complex vitamins (e.g., niacin, riboflavin, thiamine, and folic acid) needed for enzymatic and metabolic functions
  • contains nucleic acids, NAD, and other co-enzymes needed for DNA/RNA synthesis and energy metabolism
  • contains magnesium, iron, and phosphorus
• NaCl: maintains osmotic balance during bacterial growth
  • also stabilizes cell membranes and supports the function of membrane transport systems

LB Broth Recipe

• to 900 mL of distilled water, add the following to a 2 L glass flask:
  • 10.0 g Tryptone
  • 5.0 g Yeast extract
  • 10.0 g NaCl
• place a magnetic stir bar in the flask and swirl gently on a stir plate until the components are fully dissolved
• adjust the pH to 7.0 ± 0.2 (if specified)
• adjust the volume to 1.0 L using distilled water
• autoclave at 121°C for 15 minutes
• store at 4°C
• if adding antibiotics, cool to room temperature (or 4°C) before adding.

Solid Medium (slants, stabs, and plates)

Solid medium is a medium that has had agar added to it, resulting in solidification to a soft gel-like solid upon reaching room temperature. The three basic types of solid media used for bacteria are the:

• plate
• deep (or stab)
• slant

The recipe below outlines the ingredients needed to prepare a basic nutrient agar for slants, deeps, and plates. This recipe is the liquid nutrient medium altered through the addition of agar. Similarly, liquid LB broth can be modified to LB agar medium through the addition of the same amount of agar as nutrient agar. Both nutrient and LB agar can be prepared using commercially available nutrient agar powder. These powders are easy to use as they contain all necessary components in pre-measured form. To read more about these types of solid media, go to Chapter 7.2: Microbial Identification.

Lab Protocol: Preparing Agar Plates

1. Prepare the liquid media using the recipe below.
2. Autoclave to sterilize.
3. Allow to cool to to 50 to 55°C.
   • If required, add antibiotics upon reaching this temperature range.
4. Using aseptic technique, pour ~20 mL of media into sterile Petri dishes. Be careful to avoid introducing bubbles.
5. Let cool under aseptic conditions with the lid slightly ajar until fully solidified.
6. To prevent condensation dripping onto the agar surface, store the plates upside down at 4°C.

Lab Protocol: Preparing Agar Slants and Deeps

1. Prepare liquid media containing agar following Lab Protocol: Preparing Agar Plates.
2. To prepare a deep (also called a stab), dispense 5 to 7 mL of medium into sterile test tubes, using aseptic technique, cover, and allow to cool.
3. To prepare slants, dispense 5 to 7 mL of medium into sterile test tubes, using aseptic technique and cover.
   • Place tubes in a slanted position to form a slanted surface.
   • Allow to cool.

As an alternative to steps 2 and 3, liquid medium can be poured into non-sterile test tubes and then autoclaved to sterilize. The tubes can be allowed to solidify into stabs and slants. However, these preparations cannot contain antibiotics (or any other heat-sensitive compound) as autoclaving will inactivate it.

Agar Recipes

Nutrient Agar

• to 900 mL of distilled water, add the following to a 2 L glass flask:
  • 5.0 g Peptone
  • 3.0 g Beef extract
  • 5.0 g NaCl
  • 15.0 g Agar
• place a magnetic stir bar in the flask and swirl gently on a stir plate until the components are fully dissolved
• adjust the pH to 7.0 ± 0.2 (if specified)
• adjust the volume to 1.0 L using distilled water
• autoclave at 121°C for 15 minutes
• if adding antibiotics, cool the medium to 50 to 55°C before adding the antibiotic (note: higher temperature will inactivate the antibiotic; lower temperatures may allow for solidification before antibiotic addition)

LB Agar

• to 900 mL of distilled water, add the following to a 2 L glass flask:
  • 5.0 g Peptone
  • 3.0 g Beef extract
  • 5.0 g NaCl
  • 15.0 g Agar
• place a magnetic stir bar in the flask and swirl gently on a stir plate until the components are fully dissolved
• adjust the pH to 7.0 ± 0.2 (if specified)
• adjust the volume to 1.0 L using distilled water
• autoclave at 121°C for 15 minutes
• if adding antibiotics, cool the medium to 50 to 55°C before adding the antibiotic (note: higher temperature will inactivate the antibiotic; lower temperatures may allow for solidification before antibiotic addition)

Inoculating Bacterial Media

The protocols given below outline how several types of bacterial cultures can be established through inoculation. In the first protocol illustrated in Figure \(\PageIndex{1}\)., a liquid bacterial culture is used inoculate a solid bacterial medium (e.g., a slant or stab). The next protocol shown in Figure \(\PageIndex{2}\) outlines how a liquid culture can be used to "streak" an agar plate. This streak plate technique progressively thins out the bacterial load across the agar surface until individual colonies are produced from single bacterial cells (Figure \(\PageIndex{3}\)). To do this, the surface of the agar plate should be imagined as being divided into 4 equal quadrants. The bacterial sample will be successively streaked through each quadrant. The last protocol illustrated in Figure \(\PageIndex{4}\) explains how a solid bacterial culture (e.g., a slant or a plate) can be used to establish a liquid bacterial culture. The amount of time these preparations are cultured and their temperature will depend on the bacterial species and the type of bacterial preparation made. For example, liquid cultures may require a culture time of only 16 to 18 hours until sufficient cell growth is achieved, while a solid agar plate might require 24 to 36 hours bacterial colonies of significant size to appear. Because liquid cultures are often incubated "overnight" (i.e., 16 to 18 hours), they are often called an "overnight" culture.

Lab Protocol: Slant or Stab Inoculation Using a Liquid Bacterial Culture

The protocol below uses a small volume of liquid bacterial culture prepared in a test tube as the starting material. However, a larger volume of bacterial culture prepared in a flask can also be used.

1. Sterilize the loop in the flame of a Bunsen burner until red-hot. Allow to cool completely.
2. Open the test tube containing the liquid culture and sterilize the mouth of the test tube by passing it over the flame.
   • do not place the cap of the test tube on the work surface, but hold it between the little finger and the palm of the hand.
3. Dip the loop into the liquid culture.
4. Sterilize the mouth of the tube by passing it back over the flame and close the tube. Put it aside.
5. Remove the cap from the test tube containing the slant (or deep) and sterilize the mouth of the tube by passing it over the flame.
   • do not place the cap of the test tube on the work surface, hold the cap between the little finger and the palm of the hand.
6. Gently streak the loop across the top of the slant several times to transfer the bacteria from the loop to the agar.
   • if inoculating a deep (i.e., a stab), insert the inoculation loop halfway down the total depth the solid medium and pull out.
7. Incubate the prepared culture under the appropriate culture conditions.

Figure \(\PageIndex{1}\): Slant inoculation. Step 1: An inoculation loop is sterilized by flaming it in a Bunsen burner and then cooled. Step 2: The opening of a liquid bacterial culture sterilized in the flame of a Bunsen burner. Step 3: The cooled inoculation loop is inserted into the liquid culture. Step 4: The opening of the liquid culture is re-flamed, the culture tube closed, and set aside. Step 5: The opening of a slant culture tube is sterilized in the flame of a Bunsen burner. Step 6a: To prepare a slant, the inoculation loop is gently streaked over the surface of the slant several times. Step 6b: To prepare a stab, the inoculation loop is inserted halfway into the solid medium and then pulled out. The inoculation loop is re-flamed to sterilize (not shown). Step 7: The prepared culture is incubated under the appropriate conditions (e.g., 37°C for 24 hours). (Slant Inoculation by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2025))

Lab Protocol: Streaking an Agar Plate Using a Liquid Bacterial Culture

The protocol below uses a small volume of liquid bacterial culture prepared in a test tube as the starting material. However, a larger volume of bacterial culture prepared in a flask can also be used.

1. Sterilize an inoculation loop in the flame of a Bunsen burner until red-hot. Allow to cool completely.
2. Open the test tube containing the liquid culture and sterilize the mouth of the test tube by passing it over the flame.
   • do not place the cap of the test tube on the work surface, but hold it between the little finger and the palm of the hand.
3. Dip the inoculation loop into the bacterial sample.
4. Open the lid of the agar plate and place it facing up on the work surface.
5. Quickly but gently streak the loop 5 or 6 times across "quadrant 1" of the agar surface.
6. Re-flame the loop and allow it to cool completely.
7. Drag the loop from "quadrant 1" into "quadrant 2" and gently streak the loop 5 or 6 times across "quadrant 2" of the agar surface being sure to avoid the streaks made in "quadrant 1".
8. Re-flame the loop and allow it to cool completely.
9. Drag the loop from "quadrant 2" into "quadrant 3" and gently streak the loop 5 or 6 times across "quadrant 3" of the agar surface being sure to avoid the streaks made in "quadrant 2".
10. Re-flame the loop and allow it to cool completely.
11. Drag the loop from "quadrant 3" into "quadrant 4" and gently streak the loop 5 or 6 times across "quadrant 4" of the agar surface being sure to avoid the streaks made in "quadrant 3".
12. Re-flame the loop and allow it to cool completely.
13. Close the lid on the agar plate, invert it and incubate it under the appropriate culture conditions.

Figure \(\PageIndex{2}\): Streaking an agar plate (1) Sterilize the loop in the flame until red-hot. Let it cool completely. (2) Dip the sterile loop into the bacterial sample. (3) Gently streak the loop across "quadrant" 1 of the agar surface. (4) Re-flame the loop and allow it to cool. (5) Drag the loop from "quadrant" 1 into "quadrant" 2, and streak several times across the surface being careful not to touch the bacteria in "quadrant 1" (6) Re-flame the loop and allow it to cool. (7) Drag the loop from "quadrant" 2 into "quadrant" 3, and streak as outlined in step 5. (8) Re-flame the loop and allow it to cool. 9. Drag the loop from "quadrant" 3 into "quadrant" 4, and streak as outlined in step 5. (Streak Plate Procedure by Kareen Martin, CC BY 4.0)

As shown in Figure \(\PageIndex{2}\), the bacteria in "quadrant 4" have been sufficiently thinned out enough to form individual colonies.

Figure \(\PageIndex{3}\): An agar plate streaked with a mixed population of bacteria. The bacterial sample was streaked starting in "quadrant" 1 and carried over into "quadrants" 2 through 4. The location of these 4 "quadrants" is approximated with dashed black lines. The bacterial load decreases as the sample is carried into "quadrants" 3 and 4 and well-formed, individual colonies can be observed. Several individual colonies are shown in this figure (red circles). (Streak Plate by Raekwon Holden, CC BY 4.0)

Streak Plate Troubleshooting

A few common mistakes, listed below in Table \(\PageIndex{1}\), can be made while streaking an agar plate.

Table \(\PageIndex{1}\): Possible mistakes in streaked plates and their results

Mistake	Result	Reason
The inoculating loop is not sterilized in between streaking quadrants.	No isolated colonies can be seen.	Too much bacteria is added to the plate. Bacterial overgrowth.
The inoculating loop has not been cooled down and was too hot when streaking.	No growth on one or several quadrants.	Bacterial cells are dead.
The user dips the inoculation loop in the liquid culture after each quadrant streak.	No isolated colonies can be seen.	Too much bacteria is added to the plate. Bacterial overgrowth.

Lab Protocol: Inoculating a Broth Using a Solid Bacterial Culture

1. Sterilize the loop in the flame until red-hot. Allow it to cool completely.
2. Open the bacterial sample.
   • For a slant culture, hold the cap between the little finger and the palm of the hand. Sterilize the mouth of the tube by passing it over the flame.
   • For an agar plant, lift the lid up slightly from the plate.
3. Gently collect the bacterial sample.
   • Touch the loop to the bacteria growing on the surface of the slant. Close the tube.
   • Touch the loop to a bacterial colony on the agar plate. Close the plate.
4. Remove the cap from the test tube containing a small volume of culture broth and hold the cap between the little finger and the palm of the hand. Sterilize the mouth of the tube by passing it over the flame.
   • The volume of this culture broth can be a small (e.g., 2 to 5 mL) and prepared in a sterile test tube, or a larger volume prepared in a sterile flask (e.g. 5 mL to 1000 mL). Note that larger culture volumes will require longer incubation times in order to ensure the cells are in their growth phase.
5. Dip the loop into the culture broth and gently swirl to release the bacterial cells. Cap the tube.
6. Incubate the under the appropriate culture conditions.
   • Gentle shaking at 200 rpm during the incubation period can be used to increase bacterial yields.

Figure \(\PageIndex{4}\): Inoculating a liquid culture from solid media. Step 1: An inoculation loop is sterilized by flaming it in a Bunsen burner and then cooled. Step 2a: Using an agar plate, the plate is opened using aseptic techniques (not shown) and the inoculation loop is touched to a single colony. Step 2b: Using an bacterial slant, the tube is opened using aseptic techniques (not shown) and the inoculation loop is touched to the bacteria on the surface of the slant. Step 3: A liquid culture is opened an flamed using the Bunsen burner. Step 4: The inoculation loop is inserted into the liquid culture media. The tube is capped and the inoculation loop is re-flamed to sterilize (not shown). Step 5: The prepared liquid culture is incubated under the appropriate conditions (e.g., 37°C for 16 to 18 hours). (Bacterial Overnight Cultures by Patricia Zuk, CC BY 4.0; figure created in BioRender. Zuk, P. (2025))