Ch 6 Microbial Growth

Student Learning Outcomes: Classify microbes into five groups on the basis of preferred temperature range.  Explain the importance of osmotic pressure to microbial growth.  Provide a use for each of the four elements (C, N, S, P) needed in large amounts for microbial growth. Explain how microbes are classified on the basis of O2 needs. Identify ways in which aerobes avoid damage by toxic forms of O2. Describe the formation of biofilms and their potential for causing infection. Distinguish between chemically defined and complex media. Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective, differential, and enrichment media. Define colony and CFUs and describe how pure cultures can be isolated with streak plates. Explain how microbes are preserved by deep-freezing and lyophilization. Distinguish between binary fission and budding. Define generation time and explain the bacterial growth curve. Review some direct and indirect methods of measuring bacterial cell growth. 1

Microbial Growth Microbial growth: Increase in cell number, not cell size! Physical Requirements for Growth: Temperature Minimum growth temperature Optimum growth temperature Maximum growth temperature Five groups based on optimum growth temperature Psychrophiles Psychrotrophs Mesophiles Thermophiles Hyperthermophiles Psychrotrophs cause food spoilage Listeriosis is a food-borne infection that causes about 2,500 people in the United States to become ill each year, and results in about 500 deaths. It is caused by food contaminated with the bacteria Listeria monocytogenes, a rod, coccobacilli shaped bacteria that can be arranged in chains or as single cells. The bacterium is motile at 20°-25°C, and is catalase positive. It is a psychrophile; therefore, it is neither killed nor does it grow slowly at cold temperatures. Listeria monocytogenes has been isolated from several sources including: ground beef, chicken and turkey, lunch meats, hot dogs, cheese, and poorly pasteurized milk. The disease usually affects pregnant women, newborns, and the immunocompromised. Fever • Muscle aches • Diarrhea • Nausea • Sore Throat Figs. 6 .2 & 3

Figs 6.2/3: Food preservation temperatures and Effect of amount of food on its cooling rate

Physical Requirements for Growth: pH and Osmotic Pressure Most bacteria grow best between pH 6.5 and 7.5: Neutrophils Some bacteria are very tolerant of acidity or thrive in it: Acidophiles (preferred pH range 1 to 5) Molds and yeasts grow best between pH 5 and 6 Hypertonic environments (increased salt or sugar) cause plasmolysis Obligate halophiles vs. facultative halophiles Fig 6.4

Plasmolysis Fig 6.4

Chemical Requirements for Growth: C, N, S, P, etc. Carbon  Half of dry weight Chemoheterotrophs use organic carbon sources Nitrogen, Sulfur, Phosphorus Needed for ? Found in amino acids and proteins (most bacteria decompose proteins) S in thiamine and biotin Phosphate ions (PO43–) Also needed K, Mg, Ca, trace elements (as cofactors), and organic growth factors Vit B1 Vit B7

Chemical Requirements for Growth: Oxygen O2 requirements vary greatly Examples of facultative anaerobes: E.coli, yeasts etc. Many bacteria can substitute another inorganic ion for oxygen Microaerophile: Borrelia burgdorferi Helicobacter pylori Table 6.1: The Effects of Oxygen on the Growth of Various Types of Bacteria

Toxic Forms of Oxygen 2 · · Superoxide free radicals (also known as superoxide anions) Peroxide anion: O22– 2 · · Superoxide is biologically quite toxic and is deployed by the immune system to kill invading microorganisms. In phagocytes, superoxide is produced in large quantities by the enzyme NADPH oxidase for use in oxygen-dependent killing mechanisms of invading pathogens.

Fig 6.5 Biofilms Microbial communities form slime or hydrogels and share nutrients Starts via attachment of planctonic bacteria to surface structures. Sheltered from harmful factors (disinfectants etc.) Cause of most nosocomial infections Bacteria communicate by chemicals via quorum sensing (Bonnie Bassler TED conference talk) Example: Patients with indwelling catheters received contaminated heparin  Bacterial #s in contaminated heparin were too low to cause infection  84–421 days after exposure, patients developed infections Microbes adhere to surfaces and accumulate as biofilms on solid surfaces in contact with water. 2. Biofilms form on teeth, contact lenses, and catheters. 3. Microbes in biofilms are more resistant to antibiotics than are free-swimming microbes.

Culture Media Culture medium: Nutrients prepared for microbial growth Have to be sterile (not living microbes) Inoculum: Introduction of microbes into medium Culture: Microbes growing in/on culture medium Chemically defined media: Exact chemical compo- sition is known (for research purposes only) Complex media: Extracts and digests of yeasts, meat, or plants, e.g.: Nutrient broth Nutrient agar Blood agar

Agar Complex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabo- lized by microbes Liquefies at 100°C Solidifies ~40°C

Anaerobic Culture Methods Reducing media contain chemicals (e.g.: thioglycollate) that combine with O2 . Keep in tightly capped test tubes. Novel method in clinical labs for Petri plates: Add oxyrase to growth media  OxyPlate Anaerobic jars Anaerobic chambers Figs 6.6 & 6.7

Capnophiles: Aerobic Bacteria Requiring High CO2 Low O2, high CO2 conditions resemble those found in intestinal tract respiratory tract and other body tissues where pathogens grow E.g: Campylobacter jejuni Use candle jar, CO2- generator packets, or CO2 incubators Campylobacter jejuni is the most commonly reported bacterial cause of foodborne infection in the United States. Adding to the human and economic costs are chronic sequelae associated with C. jejuni infection—Guillian-Barré syndrome and reactive arthritis. In addition, an increasing proportion of human infections caused by C. jejuni are resistant to antimicrobial therapy. Mishandling of raw poultry and consumption of undercooked poultry are the major risk factors for human campylobacteriosis. Efforts to prevent human illness are needed throughout each link in the food chain. Photograph: Scanning electron microscope image of Campylobacter jejuni, illustrating its corkscrew appearance and bipolar flagella. Source: Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia. Candle jar

Selective Media and Differential Media Selective medium: Additives suppress unwanted and encourage desired microbes – e.g. EMB, mannitol salt agar etc. Differential medium: changed in recognizable manner by some bacteria  Make it easy to distinguish colonies of different microbes – e.g.  and  hemolysis on blood agar; MacConkey agar, EMB, mannitol salt agar etc. Compare to Fig 6.9

EMB MacConkey

MSA Fig 6.10

Enrichment Media/Culture Encourages growth of desired microbe Example: Assume soil sample contains a few phenol- degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil and incubate Transfer 1 ml to another flask of the phenol medium and incubate Only phenol-metabolizing bacteria will be growing

Pure Cultures Contain only one species or strain. Most patient specimens and environmental samples contain several different kinds of bacteria Streak-plate method is commonly used. More details in lab. Colony formation: A population of cells arising from a single cell or spore or from a group of attached cells (also referred to as CFU). Only ~1% of all bacteria can be successfully cultured Aseptic technique critical! Compare to Fig. 6.11

Preserving Bacterial Cultures Deep-freezing: Rapid cooling of pure culture in suspension liquid to –50°to –95°C. Good for several years. Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum. Good for many years. Regular freezer -20 degrees Celcius Refco – 80 degree Celcius

The Growth of Microbial Cultures Binary fission – exponential growth Budding Generation time – time required for cell to divide (also known as doubling time) Ranges from 20 min (________) to > 24h (_____________) Consider reproductive potential of E. coli Fig 6.12a

Fig 6.13 Figure 6.12b

Bacterial Growth Curve Foundation Fig 6.15 Illustrates the dynamics of growth Phases of growth Lag phase Exponential or logarithmic (log) phase Stationary phase Death phase (decline phase) Compare growth in liquid and on solid media

Direct Measurements of Microbial Growth Viable cell counts: Plate counts: Serial dilutions put on plates CFUs form colonies Measuring cell products: Acid production Gas production ATP generation Fig 6.16

Fig 6.17 Figure 6.15, step 1

Additional Direct Counts Direct microscopic count: Counting chambers (slides) for microscope Filtration method of choice for low counts Fig 6.18

Indirect Count: Spectrophotometry Measures turbidity OD (Absorbance) is function of cell number Fig 6.21 The End