Microbial Nutrition, Ecology, and Growth Chapter 7 Copyright © The McGraw-Hill Companies, Inc) Permission required for reproduction or display.

Learning Objectives: Classify microbes into five groups on the basis of preferred temperature range. Identify how and why the pH of culture media is controlled. Explain the importance of osmotic pressure to microbial growth. Explain how microbes are classified on the basis of oxygen requirements. Identify ways in which aerobes avoid damage by toxic forms of oxygen

Learning Objectives: Define bacterial growth, including binary fission. Compare the phases of microbial growth and describe their relation to generation time. Describe three direct methods for measuring microbial growth. Differentiate between direct and indirect methods for measuring cell growth. Explain three indirect methods of measuring cell growth

Temperature Optima 505 Minimum Temperature °C Maximum Optimum Psychrophile Psychrotroph Thermophile Mesophile Extreme thermophile Rate of Growth Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Psychrotrophs Grow between 0°C and 20°C Cause food spoilage

pH Optima Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Acidophiles grow in acidic environments Alcaliphiles grow in basic environments acid spring water 2.3 lemon juice 2.4 vinegar 3.0 red wine 3.5 sauekraut 4.2 beer 4.6 acid rain 5.0 cheese yogurt 6.6 cow’s milk 7.0 distilled water 7.4 human blood 8.0 seawater 8.4 sodium bicarbonate9.2 borax, alkaline soils 10.5 milk of magnesia 11.5 household ammonia 12.4 limewater 0pH 13.2 oven cleaner 1M potassium hydroxide 0.1 M hydrochloric acid Acidic[H + ] Neutral[OH – ] Basic (alkaline)

Plasmolysis Osmotic pressure Hypertonic environments, increase salt or sugar, cause plasmolysis Extreme or obligate halophiles require high osmotic pressure Facultative halophiles tolerate high osmotic pressure

Conclusions: On the basis of preferred temperature range, microbes are classified as psychrophiles cold-loving), mesophiles (moderate-temperature-loving), and thermophiles (heat- loving). The minimum growth temperature is the lowest temperature at which a species will grow, the optimum growth temperature – at which it grows best, and the maximum temperature – the highest at which growth is possible. Most bacteria grow best at a pH value between 6.5 and 7.5 In a hypertonic solution, most microbes undergo plasmolysis; halophiles can tolerate high salt concentrations

Oxygen Requirements Oxygen is needed for aerobic respiration. Oxygen is a powerful oxidizing agent (toxic to cells) Some microbes use oxygen and can detoxify it. Some microbes do not use oxygen and cannot detoxify it. Some microbe do not use oxygen but can detoxify it.

Toxic Oxygen Species Singlet oxygen: O 2 boosted to a higher-energy state Superoxide free radicals: O 2 – Peroxide anion: O 2 2– Hydroxyl radical (OH  )

Microbe Requirements for Growth Table 6.1 Obligate Aerobes Facultative Anaerobes Obligate Anaerobes Aerotolerant Anaerobes Micro- aerophiles Require O 2 Does not require O 2 No O 2 Can survive in presence of O 2 Require low concentration of O 2

Oxygen Requirement in Thioglycollate Broth Aerobes Microaerophiles Anaerobes Facultative anaerobes Aerotolerant anaerobe

Anaerobic Culture Methods Anaerobic jar serves the purpose of chemically removing oxygen Figure 6.5

Capnophiles Require High CO 2 Candle jar can be used to grow Neissseria meningitidis CO 2 -packet is used to generate an environment that contains more carbon dioxide than oxygen Figure 6.7

Other Factors Barometric pressure - barophiles Dry - Xerophiles

Conclusions: On the basis of oxygen requirements, organisms are classified as obligate aerobes, facultative anaerobes, obligate anaerobes, aerotolerant anaerobes, and microaerophiles. Aerobes, facultative anaerobes, and aerotolerant anaerobes must have the enzymes superoxide dismutase, and either catalase or peroxidase.

Bacterial Reproduction Binary fission Asexual process Doubling time (generation) if 20 minutes, then in 24 hours 1  4.7 x cells 5,100 tons Ribosomes Cell wall Cell membrane Chromosome 1 Chromosome 2 When septum is complete, cells are considered divided. Some species will separate completely as shown here, while others remain attached, Septum formation begins. Protein band forms in center of cell. Chromosome is replicated and new and old chromosomes move to different sides of cell. A young cell.

The Math of Bacterial Growth X = X 0 * 2 Y (a) Number Time ,000 10,000 10,000,000, (b) Number of generations Exponential value (2 × 1) (2 × 2)(2 × 2 × 2) (2 × 2 × 2 × 2) (2 × 2 × 2 × 2 × 2) () () Number of cells Log of number of cells Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Bacterial Growth Curve Implications for: Applying antimicrobial agents Treating infections

Conclusions: The normal reproductive method of bacteria is binary fission, in which a single cell divides into two identical cells. The time required for a cell to divide or a population to double is the generation time. Bacterial division occurs according to a logarithmic progression. During the lag phase, there is little or no change in the number of cells, but metabolic activity is high. During the log phase, the bacteria multiply at a fastest rate possible under the conditions provided. During the stationary phase, there is an equillibrium between cell division and death During the death phase, the number of deaths exceeds the number of new cells formed.

Measuring Microbial Growth Direct methods Plate counts Filtration Direct microscopic count Automated cell count Indirect methods Turbidity Metabolic activity Dry weight

Plate Count After incubation, count colonies on plates that have colonies (CFUs)

Plate Count Method Inoculate Petri plates from serial dilutions using either method Incubate plates and count up the number of colonies Figure 6.16

Direct Measurements of Microbial Growth Filtration

Direct Cell Counts Cytometer Known volume Count total cells Both dead and live cells are counted

Direct Measurements of Microbial Growth Direct microscopic count

Automatic counter Sample in liquid Bacterial cell Tube Counting orifice Electronic detector Automated Cell Counting Coulter counter Flow cytometer Can sort cells Requires tagging Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Bacteria Scatter Light Spectrophotometer Transmittance Absorbance Red light scatters best Percentage of light transmitted High Low (2) (b) (a) (1) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. a: © Kathy Park Talaro/Visuals Unlimited.

Measure Cell Components We can measure growth by increase in mass… Measure dry weight of cells Assume that relative fraction of the cell components is relatively stable Lipid content DNA content Protein determinations (most common)

Conclusions: A standard plate count reflects the number of viable microbes and assumes that each bacterium grows into a single colony; it is reported as the number of colony forming units (CFU) In filtration, bacteria are retained on the surface of a membrane filter and then transferred to a culture medium to grow and to be counted. In a direct count, the microbes in a measured volume of a bacterial suspension are counted with the use of a specially designed slide.

Conclusions: A spectrophotometer is used to determine turbidity by measuring the amount of light that passes through a suspension of cells. An indirect way of estimating bacterial numbers is measuring the metabolic activity of a population (for example, acid production or oxygen consumption)