LAB 16

Measurement of Bacterial Growth Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves increase in cell mass and number of ribosome, duplication of the bacterial chromosome, synthesis of new cell wall and plasma membrane, partitioning of the two chromosomes, septum formation, and cell division. This asexual process of reproduction is called binary fission.

Growth in Batch Culture “Growth” is generally used to refer to the acquisition of biomass leading to cell division, or reproduction A “Batch culture” is a closed system in broth medium in which no additional nutrient is added after inoculation of the broth. “Generation time” Time taken for a cell population to double in numbers and thus equivalent to the average length of the cell cycle

The Bacterial Growth Curve Four characteristic phases of the growth cycle are recognized. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.

The length of the lag phase is apparently dependent on a wide variety of factors including: The size of the inoculum. Time necessary to recover from physical damage or shock in the transfer. Time required for synthesis of essential coenzymes or division factors. Time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.

Exponential (log) Phase Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is expressed as generation time, also the doubling time of the bacterial population.

Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: Exhaustion of available nutrients. Accumulation of inhibitory metabolites or end products Exhaustion of space, in this case called a lack of "biological space". Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric measurements or microscopic counts, the death phase cannot be observed.). During the death phase, the number of viable cells decreases geometrically (exponentially), essentially the reverse of growth during the log phase

Growth in Continuous Culture A “continuous culture” is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat. Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained.

Factors that Influence Growth Temperature. pH. Salt concentration. Oxygen concentration.

Turbidity measurements of microbial growth Optical Density (Counting by Spectrophotometer)

Materials TSB of Nonpathogenic Escherichia coli Inoculating loop Spectrophotometer Test tube rack 2 tubes of TSB 37C Incubator Bunson burner Surface disinfectant

Procedure Wash hands and put on gloves. Assemble equipment and materials and prepare work area. Set the spectrophotometer at 450nm and let it warm up 30 minutes before performing the readings. The meter on the spectrophotometer should read “0% Transmittance”. Obtain 2 tubes of TSB. Label one C (uninoculated) for control and one S (inoculated) for sample. Place tube C into the sample holder of the spectrophotometer. Turn the light control knob until the meter needle is on “100% Transmittance”. Remove the control tube. The spectrophotometer is now standardized.

Flame and cool the loop. Mix the culture of E. coli and inoculate tube S with 5 loopfuls of the broth. Flame and cool the loop. Start the turbidity readings by placing the S tube into the sample well and read the OD (absorbance). Record the results in the data section. Take OD reading over the time period specified in the data section and return the TSB tube to the 37 C incubator between each reading. Turn off the spectrophotometer upon completion of the readings. Plot the readings on the graph paper as absorbance vs. time. Clean work area with disinfectant. Remove gloves and wash hands with disinfectant.

Record the absorbance value for each sample reading starting at 0 time. Plot the absorbance values vs. time on graph paper. Label the x and y axis. Each of the readings will represent the growth of the E. coli in the culture. Label each part of the growth curve: Lag phase, Exponential growth phase, and the Stationery phase.

Microbial Control Agents Many microorganisms are beneficial and necessary for human well-being. However, microbial activities may have undesirable consequences, such as food spoilage and disease. It is essential to be able to kill microorganisms or inhibit their growth to minimize their destructive effects. Physical, chemical, and mechanical methods to destroy or reduce undesirable microbes in a given area

Radiation Radiation is example for Physical Methods of Microbial Control. Types of Radiation:- Ionizing radiation. Nonionizing radiation.

Ionizing radiation: deep penetrating power that has sufficient energy to cause electrons to leave their orbit, breaks DNA. E.g. Gamma rays, X-rays, Cathode rays. Used to sterilize medical supplies and food products.

Nonionizing radiation: have little penetrating power and must be directly exposed. UV light creates thymine dimers, which interfere with replication.

Terminology of Microbial Control Sterilization: Killing or removing all forms of microbial life (including endospores) in a material or an object. Disinfection: are chemicals used on nonliving surfaces to inhibit the growth of microorganisms. Antiseptic: are chemicals used on living tissues to inhibit the growth of microorganisms. Chemotherapeutic agents (antibiotics): are chemicals used to destroy or inhibit the growth of microorganisms in living tissues.

Antimicrobial Chemotherapy Antimicrobial Chemotherapy : is the use of chemicals to inhibit or kill microorganisms in or on the host. Based on their origin, there are 2 general classes of antimicrobial chemotherapeutic agents: Antibiotics: substances produced as metabolic products of one microorganism which inhibit or kill other microorganisms. Antimicrobial Chemotherapeutic Chemicals: chemicals synthesized in the laboratory which can be used therapeutically on microorganisms.

Antimicrobial Agents Antimicrobial agents are : Cidal in action: they kill microorganisms (e.g., penicillins, cephalosporins, streptomycin, neomycin). Static in action : they inhibit microbial growth long enough for the body's own defenses to remove the organisms (e.g., tetracyclines, erythromycin, sulfonamides). Selective Toxicity: This means that the agent used must inhibit or kill the microorganism without seriously harming the host.

Antimicrobial agents also vary in their spectrum: Broad spectrum : Drugs which are effective against a variety of both gram-positive and gram-negative bacteria : (e.g., tetracycline, streptomycin, cephalosporins, ampicillin, sulfonamides). Narrow spectrum : Drugs which are effective against just gram-positive bacteria, just gram negative bacteria, or only a few species are termed (e.g., penicillin G, erythromycin, clindamycin, gentamicin). If a choice is available, a narrow spectrum is preferable since it will cause less destruction to the body's normal flora. In fact, indiscriminate use of broad spectrum antibiotics can lead to super infection by opportunistic microorganisms.

Antimicrobial Drugs Antibiotic Resistance: bacteria gain ability to grow; no longer sensitive to drug Antiretroviral: act specifically against viruses Combination of drugs: Synergism: action of two antibiotics greater. Antagonism: action of drug is reduced; less effective. Minimum Inhibitory Concentration (MIC):The lowest concentration of chemotherapeutic agent capable of preventing growth of the test organism. Minimum Bactericidal Concentration (MBC): MBC is the lowest concentration of the chemotherapeutic agent that results in no growth (turbidity) of the subcultures.

Antimicrobial Activity Five Modes of Antimicrobial Activity: Injury to Plasma Membrane (polymixin B). Inhibition of Cell Wall Synthesis (penicillins, bacitracin). Inhibition of Protein Synthesis (translation). Inhibition of Nucleic Acid replication & transcription. Inhibition of essential metabolites.

Haw To Choose Antimicrobial Drugs? Narrow spectrum OR broad spectrum. Old age OR child. Male OR female. Pregnant OR lactating women. In patient OR out patient. Type of microorganisms. Site of infection.

END OF LECTURE