Question

Select 3 of the factors that affect microbial growth and discuss. (USLO 4.1)

Discuss microbial growth patterns. (USLO 4.2)

Discuss the different culture methods used in microbiology. (USLO 4.3)

Identify 2 growth control mechanisms used to control microbial growth. Explain how each works. (USLO 4.4)

Correlate aseptic technique in healthcare and microbiology. (USLO 4.5)

Answer 1

1) 3 of the factors that affect microbial growth are:

Moisture:

Water is an essential component for the growth of the bacteria. Therefore, the presence of a free water molecule is important for the optimum growth of the microorganism. Apart from that desiccation or drying has a severe effect on microbes. For example Treponema pallidum, N. gonorrhoeae can die easily due to desiccation. While bacterial pathogens like M. tuberculosis, S. aureus can survive desiccation for several weeks. Therefore, it is understood the required moisture content buys the microbe varies species to species. Hence, the moisture content needs to be maintained for optimum growth.

Temperature:

It is an essential environmental factor that can influence the growth of the organisms. Most of the pathogens grow at 37 ⁰ C (body temperature). Bacteria are categorized under three groups on the basis of the optimum temperature range

• Mesophile: The optimum temperature range for mesophiles is 25⁰C to 40⁰ Most of the pathogenic bacteria come under this group.
• Psychrophile: The optimum temperature for psychrophiles is below 20⁰
• Thermophile: The optimum temperature range for thermophiles is 55⁰C to 80⁰ Eg: Bacillus stearothermophilus.

pH:

pH is an essential factor for the growth of the microbes. May bacteria are able to produce several organic acids which reduce the pH of the medium and also restrict the growth of other bacteria. Apart from that some media constituents can be affected by low pH. Therefore, maintain the optimum pH is highly important to obtain adequate growth of the organisms. Pathogens are mostly requiring neutral pH (7.2).

2) Microbial growth patterns are: Provided with the right conditions (food, correct temperature, etc) microbes can grow very quickly. Bacteria and archaea reproduce asexually only, while eukartyotic microbes can engage in either sexual or asexual reproduction. Bacteria and archaea most commonly engage in a process known as binary fission, where a single cell splits into two equally sized cells. Other, less common processes can include multiple fission, budding, and the production of spores.

The bacterial growth curve represents the number of live cells in a bacterial population over a period of time.

• Lag Phase: This initial phase is characterized by cellular activity but not growth. A small group of cells are placed in a nutrient rich medium that allows them to synthesize proteins and other molecules necessary for replication. These cells increase in size, but no cell division occurs in the phase.
• Exponential (Log) Phase: After the lag phase, bacterial cells enter the exponential or log phase. This is the time when the cells are dividing by binary fission and doubling in numbers after each generation time. Metabolic activity is high as DNA, RNA, cell wall components, and other substances necessary for growth are generated for division. It is in this growth phase that antibiotics and disinfectants are most effective as these substances typically target bacteria cell walls or the protein synthesis processes of DNA transcription and RNA translation.
• Stationary Phase: Eventually, the population growth experienced in the log phase begins to decline as the available nutrients become depleted and waste products start to accumulate. Bacterial cell growth reaches a plateau, or stationary phase, where the number of dividing cells equal the number of dying cells. This results in no overall population growth. Under the less favorable conditions, competition for nutrients increases and the cells become less metabolically active. Spore forming bacteria produce endospores in this phase and pathogenic bacteria begin to generate substances (virulence factors) that help them survive harsh conditions and consequently cause disease.
• Death Phase: As nutrients become less available and waste products increase, the number of dying cells continues to rise. In the death phase, the number of living cells decreases exponentially and population growth experiences a sharp decline. As dying cells lyse or break open, they spill their contents into the environment making these nutrients available to other bacteria. This helps spore producing bacteria to survive long enough for spore production. Spores are able to survive the harsh conditions of the death phase and become growing bacteria when placed in an environment that supports life.

3) Different culture methods used in microbiology are:

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Microbial cultures are foundational and basic diagnostic methods used as a research tool in molecular biology.

a) Bacterial culture:

There are several types of bacterial culture methods that are selected based on the agent being cultured and the downstream use.

Broth cultures: One method of bacterial culture is liquid culture, in which the desired bacteria are suspended in a liquid nutrient medium, such as Luria Broth, in an upright flask. This allows a scientist to grow up large amounts of bacteria for a variety of downstream applications.

Agar plates: Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the optimal temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius, or the human body temperature, for cultures from humans or animals, or lower for environmental cultures). After the desired level of growth is achieved, agar plates can be stored upside down in a refrigerator for an extended period of time to keep bacteria for future experiments.

Stab cultures: Stab cultures are similar to agar plates, but are formed by solid agar in a test tube. Bacteria is introduced via an inoculation needle or a pipette tip being stabbed into the center of the agar. Bacteria grow in the punctured area. Stab cultures are most commonly used for short-term storage or shipment of cultures.

Solid plate culture of thermophilic microorganisms: For solid plate cultures of thermophilic microorganisms such as Bacillus acidocaldarius, Bacillus stearothermophilus, Thermus aquaticus and Thermus thermophilus etc. growing at temperatures of 50 to 70 degrees C, low acyl clarified gellan gum has been proven to be the preferred gelling agent comparing to agar for the counting or isolation or both of the above thermophilic bacteria.

b) Virus and phage culture:

Virus or phage cultures require host cells in which the virus or phage multiply. For bacteriophages, cultures are grown by infecting bacterial cells. The phage can then be isolated from the resulting plaques in a lawn of bacteria on a plate. Virus cultures are obtained from their appropriate eukaryotic host cells.

c) Eukaryotic cell culture:

Isolation of pure cultures: For single-celled eukaryotes, such as yeast, the isolation of pure cultures uses the same techniques as for bacterial cultures. Pure cultures of multicellular organisms are often more easily isolated by simply picking out a single individual to initiate a culture. This is a useful technique for pure culture of fungi, multicellular algae, and small metazoa, for example.

4) 2 growth control mechanisms used to control microbial growth are:

Autoclaving (steam under pressure or pressure cooker): Autoclaving is the most effective and most efficient means of sterilization. All autoclaves operate on a time/temperature relationship. These two variables are extremely important. Higher temperatures ensure more rapid killing. The usual standard temperature/pressure employed is 121ºC/15 psi for 15 minutes. Longer times are needed for larger loads, large volumes of liquid, and more dense materials. Autoclaving is ideal for sterilizing biohazardous waste, surgical dressings, glassware, many types of microbiologic media, liquids, and many other things. However, certain items, such as plastics and certain medical instruments (e.g. fiber-optic endoscopes), cannot withstand autoclaving and should be sterilized with chemical or gas sterilants. When proper conditions and time are employed, no living organisms will survive a trip through an autoclave.

High pressures enable steam to reach high temperatures, thus increasing its heat content and killing power. Most of the heating power of steam comes from its latent heat of vaporization. This is the amount of heat required to convert boiling water to steam. This amount of heat is large compared to that required to make water hot. For example, it takes 80 calories to make 1 liter of water boil, but 540 calories to convert that boiling water to steam. Therefore, steam at 100º C has almost seven times more heat than boiling water.

Moist heat is thought to kill microorganisms by causing denaturation of essential proteins. Death rate is directly proportional to the concentration of microorganisms at any given time. The time required to kill a known population of microorganisms in a specific suspension at a particular temperature is referred to as thermal death time (TDT). Increasing the temperature decreases TDT, and lowering the temperature increases TDT. Processes conducted at high temperatures for short periods of time are preferred over lower temperatures for longer times.

Irradiation: usually destroys or distorts nucleic acids. Ultraviolet light is commonly used to sterilize the surfaces of objects, although x-rays, gamma radiation and electron beam radiation are also used.

Ultraviolet lamps are used to sterilize workspaces and tools used in microbiology laboratories and health care facilities. UV light at germicidal wavelengths (two peaks, 185 nm and 265 nm) causes adjacent thymine molecules on DNA to dimerize, thereby inhibiting DNA replication (even though the organism may not be killed outright, it will not be able to reproduce). However, since microorganisms can be shielded from ultraviolet light in fissures, cracks and shaded areas, UV lamps should only be used as a supplement to other sterilization techniques.

Gamma radiation and electron beam radiation are forms of ionizing radiation used primarily in the health care industry. Gamma rays, emitted from cobalt-60, are similar in many ways to microwaves and x-rays. Gamma rays delivered during sterilization break chemical bonds by interacting with the electrons of atomic constituents. Gamma rays are highly effective in killing microorganisms and do not leave residues or have sufficient energy to impart radioactivity.

Electron beam (e-beam) radiation, a form of ionizing energy, is generally characterized by low penetration and high-dose rates. E-beam irradiation is similar to gamma radiation in that it alters various chemical and molecular bonds on contact. Beams produced for e-beam sterilization are concentrated, highly-charged streams of electrons generated by the acceleration and conversion of electricity.

e-beam and gamma radiation are for sterilization of items ranging from syringes to cardiothoracic devices.

5) Aseptic technique in healthcare and microbiology are:

Barriers: Barriers protect from the transfer of pathogens. Some barriers used in aseptic technique include

• sterile gloves
• sterile gowns
• masks
• sterile drapes

Sterile barriers are those that have not touched a contaminated surface. They’re specially packaged and cleaned items. Healthcare workers put them on or use them in specific ways that minimize exposure to germs.

Equipment Preparation: Healthcare providers also use sterile equipment and sterile instruments. To further protect the patient, they apply cleansing and bacteria-killing preparations.

Environmental controls: Maintaining a sterile environment requires keeping doors closed during an operation. Only necessary health personnel should be at the procedure. The more people present, the more opportunities for harmful bacteria to cause contamination.

Contact guidelines: Once healthcare providers have on sterile barriers, they should only touch other sterile items. They should avoid touching nonsterile items at all costs.