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 2 ways in which aerobes avoid damage by toxic forms of O2. Explain biofilms, describe their formation 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 and differential 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

SLOs cont.: Check Your Understanding How would one determine whether a microbe is a strict anaerobe? If bacterial cells were given a sulfur source containing radioactive sulfur (35S) in their culture media, in what molecules would the 35S be found in the cells?

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 Fig 6.1

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, the so-called ______________ , grow best between pH 6.5 and 7.5. Some bacteria prefer a pH range 1 to 5. These are the __________________ What bacterium lives in the stomach? Some bacteria do well in hypertonic (?) environments: Obligate vs. facultative __________________

What happens if bacterium is not salt tolerant? Fig 6.4

Chemical Requirements for Growth: C, N, S, P, etc. Carbon  Half of dry weight Chemoheterotrophs use organic carbon sources Nitrogen and Sulfur Found in ? Needed for ? S also in thiamine and biotin Phosphorus: 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 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 · ·

Fig 6.5 Biofilms Microbial communities form slime or hydrogels and share nutrients 1st step: Attachment of planctonic bacteria to surface structures. Bacteria in biofilm are sheltered from harmful factors (disinfectants etc.) Cause of most nosocomial infections, i.e.: : Indwelling catheters Bacteria communicate by chemicals via quorum sensing “The secret social lives of bacteria” TED talk by Bonnie Bassler https://www.youtube.com/watch?v=TVfmUfr8VPA

Culture Media Culture medium: Nutrients prepared for microbial growth Have to be sterile (?) 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 grow better in high ____________ and low ______________ In human body, these conditions found in __________________ E.g: Campylobacter jejuni Use candle jar, CO2- generator packets, or CO2 incubators Candle jar

Biosafety Levels Biosafety Level 3 https://www.youtube.com/watch?v=DLypons50g4 Biosafety Level 4 https://www.youtube.com/watch?v=Hlqic9RP7sI

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

EMB MacConkey

MSA Compare to Fig 6.10

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.

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 (_____________) Fig 6.12a

Consider reproductive potential of E. coli! Fig 6.13 Type of curve? Fig 6.14

Bacterial Growth Curve Foundation Fig 6.15 Illustrates the dynamics of growth Phases of growth ________ phase Exponential or logarithmic (_____) phase _______________ phase _____________ 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 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 Fig 6.21 Measures turbidity OD (Absorbance) is function of cell number

Clinical Case: Glowing in the Dark In the Clinic H2O2 and wound care Clinical Case: Glowing in the Dark The End