Growth at 42°C Test- Principle, Procedure, Result, Uses

The growth at 42°C test is a simple and rapid test that can help in the identification of some bacteria based on their ability to grow at a high temperature. The test is especially useful for the differentiation of Pseudomonas aeruginosa from other fluorescent pseudomonads, as well as other non-fermentative bacteria. Pseudomonas aeruginosa is a Gram-negative rod that can cause serious infections in humans, especially in immunocompromised patients and those with cystic fibrosis. The organism is also known for its resistance to many antibiotics and disinfectants, making it difficult to treat and control. Therefore, it is important to identify P. aeruginosa accurately and promptly in clinical specimens.

The growth at 42°C test has two main objectives:

• To differentiate Pseudomonas aeruginosa from the other fluorescent Pseudomonads. P. aeruginosa is a clinically significant pathogen that can cause serious infections in humans and animals. It is also resistant to many antibiotics and disinfectants. Therefore, it is important to identify and isolate this organism from clinical and environmental samples.
• To differentiate between other non-fermentative bacteria. Non-fermentative bacteria are a diverse group of Gram-negative bacteria that do not ferment carbohydrates. They are often found in soil, water, and hospital environments. Some of them can cause opportunistic infections in immunocompromised or debilitated patients. Examples of non-fermentative bacteria include Acinetobacter, Burkholderia, Stenotrophomonas, and Alcaligenes.

The growth at 42°C test is based on the ability of an organism to survive and grow at a temperature of 42°C, which is higher than the normal body temperature of most mammals. This ability allows the differentiation of P. aeruginosa from the other fluorescent pseudomonads, which cannot grow at this temperature. The test also helps to distinguish non-fermentative bacteria from other Gram-negative bacteria that can grow at 37°C but not at 42°C. The test is simple, rapid, and inexpensive to perform. It can be used as a screening test for the presumptive identification of P. aeruginosa and other non-fermentative bacteria. However, it should be confirmed by other biochemical or molecular tests for a definitive diagnosis.

The growth at 42°C test is mainly used to test Gram-negative aerobic rods that are presumptive of Pseudomonas aeruginosa. P. aeruginosa is a clinically significant pathogen that can cause a variety of infections, especially in immunocompromised patients. It is also resistant to many antibiotics and disinfectants, making it difficult to treat and control.

P. aeruginosa belongs to the fluorescent pseudomonads group, which are characterized by their ability to produce pigments that fluoresce under ultraviolet light. However, not all strains of P. aeruginosa produce pigments, and some non-pigmented strains can be confused with other fluorescent pseudomonads.

The growth at 42°C test helps to differentiate P. aeruginosa from other fluorescent pseudomonads, such as Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas stutzeri. These species can grow at 37°C but not at 42°C, whereas P. aeruginosa can grow at both temperatures.

The test can also be used to differentiate other non-fermentative bacteria that can grow at 42°C, such as Acinetobacter baumannii, Burkholderia cepacia, and Stenotrophomonas maltophilia. These bacteria are also opportunistic pathogens that can cause nosocomial infections.

The test is not suitable for testing Gram-positive bacteria or anaerobic bacteria, as they cannot grow on the medium or at the temperature used for the test.

Media Used

Tryptic soya Agar is used for the cultivation of fastidious organisms like Salmonella, Pseudomonas, Listeria, etc. It is thus used for the testing of the ability of an organism to grow at 42°C. The composition of the media is given below:

Reagents Used

No reagents are required for this test.

Supplies Used

• Incubator at 37°C and 42°C
• Inoculating loop or needle
• Sterile tryptic soya agar slants
• Bacterial cultures to be tested

The procedure of the growth at 42°C test is as follows:

1. Prepare the tryptic soya agar medium by dissolving 45 grams of the dehydrated powder in 1000 milliliters of distilled water. Heat the solution to boiling and sterilize it in an autoclave at 121°C for 15 minutes.
2. Pour the sterilized medium into sterile tubes and cool them at a slanted position to form agar slants with butts of 1.5 – 2.0 cm depth.
3. Inoculate the agar slants with a light inoculum of the test organism by lightly touching the middle of a single 18-24 hour colony with an inoculating loop or needle. Streak the surface of the slant with the inoculum.
4. Incubate one tube at 37°C and another tube at 42°C for 24 hours in an incubator.
5. Observe the tubes for growth after incubation. Compare the growth at both temperatures and record the result.

The procedure is simple and can be performed in any microbiology laboratory with basic equipment and supplies. The test is inexpensive and rapid, and can provide a presumptive identification of Pseudomonas aeruginosa and other non-fermentative bacteria. However, it should be noted that the test is not confirmatory and should be followed by other tests for definitive identification.

The result of the growth at 42°C test is based on the observation of growth on the agar slants after incubation at 37°C and 42°C for 24 hours. The growth is indicated by the presence of visible colonies on the surface of the agar. The result can be interpreted as follows:

• Positive result: Growth at both 37°C and 42°C indicates a positive result. This means that the organism can survive and grow at a high temperature of 42°C. This is a characteristic feature of Pseudomonas aeruginosa, which differentiates it from other fluorescent pseudomonads and non-fermentative bacteria.
• Negative result: Growth at 37°C with no growth at 42°C indicates a negative result. This means that the organism cannot survive or grow at a high temperature of 42°C. This is a characteristic feature of other fluorescent pseudomonads and non-fermentative bacteria that are not P. aeruginosa.

The result interpretation should be confirmed by other biochemical tests, such as acetamide utilization test, oxidase test, pyocyanin production test, etc., to identify the organism accurately.

Control organisms are used to check the validity and accuracy of the test. They are organisms that are known to give a positive or negative result for the test. For the growth at 42°C test, the following control organisms are used:

• Positive: Pseudomonas aeruginosa is a Gram-negative rod that can grow at both 37°C and 42°C. It is the most common species of Pseudomonas that causes infections in humans and animals. It produces a blue-green pigment called pyocyanin and a grape-like odor.
• Negative: Pseudomonas fluorescens is a Gram-negative rod that can grow at 37°C but not at 42°C. It is a common soil and water bacterium that produces a yellow-green fluorescent pigment. It is usually harmless to humans and animals, but can cause spoilage of food and dairy products.

The control organisms are inoculated on the same medium and incubated at the same conditions as the test organism. The results of the control organisms should match the expected results, otherwise the test is invalid and should be repeated.

The growth at 42°C test is a simple and rapid test that can be used for the presumptive identification of Pseudomonas aeruginosa and other non-fermentative bacteria. The test has the following uses:

• The test can help to differentiate apyocyanogenic (non-pigmented) P. aeruginosa from other fluorescent pseudomonads. Apyocyanogenic P. aeruginosa can cause serious infections in humans and animals, especially in immunocompromised patients. The test can thus aid in the early diagnosis and treatment of these infections.
• The test can also help to differentiate species of the fluorescent pseudomonas group, such as P. fluorescens, P. putida, P. stutzeri, etc. These species have different ecological and clinical significance and can be involved in various biotechnological processes, such as bioremediation, biocontrol, and biodegradation.
• The test can also help to differentiate other non-fermentative bacteria, such as Acinetobacter, Alcaligenes, Burkholderia, etc. These bacteria can also cause opportunistic infections in humans and animals and have different antibiotic susceptibility patterns. The test can thus help to guide the appropriate antimicrobial therapy.

The growth at 42°C test is a useful screening test that can provide preliminary information about the identity of an organism. However, it should not be used as a sole criterion for identification and should be confirmed by other biochemical or molecular tests.

• The test is not a definitive identification method for Pseudomonas aeruginosa, as some strains of other fluorescent pseudomonads may also grow at 42°C. Therefore, additional tests such as oxidase test, pigment production, and acetamide utilization should be performed to confirm the identity of the organism.
• The test may give false-negative results if the inoculum is too heavy or if the incubation time is too short. A light inoculum and a minimum of 24 hours of incubation are recommended for optimal results.
• The test may give variable results depending on the type and quality of the medium used. Tryptic soya agar is the recommended medium for this test, as it supports the growth of most non-fermentative bacteria. Other media such as nutrient agar or MacConkey agar may not be suitable for this test, as they may inhibit the growth of some organisms or allow the growth of others that are not normally able to grow at 42°C.
• The test may be affected by environmental factors such as humidity, oxygen concentration, and pH. These factors should be controlled and monitored during the incubation period to ensure accurate results.

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