Microbial Growth and Nutrition Nestor T. Hilvano, M.D., M.P.H. Images Copyright by Bauman, Robert Microbiology, With Diseases by Taxonomy, 3rd edition, Pearson Benjamin Cummings

Learning Objectives You should be able to: 1.Compare the four basic categories of organisms based on their carbon and energy sources. 2.Distinguish among anaerobes, aerobes, aerotolerant anaerobes, facultative anaerobes, and microaerophiles. 3.Explain how extremes of temperature, pH, and osmotic pressure limit microbial growth. 4.Describe methods for collecting clinical specimens. 5.Describe the two most common methods by which microorganisms can be isolated for culture. 6.Describe six types of general culture media available for bacterial culture. 7.Explain what is meant by the generation time of bacteria. 8.Draw, label, and describe a bacterial growth curve. 9.Contrast viable plate count and turbidity methods of measuring bacterial growth.

Microbial Growth Requirements Chemical 1. Nutrients – C,O, N, H; source of carbon, energy, and electrons or hydrogen atoms 2. Trace elements and organic materials, growth factors Physical – temperature, pH, osmolarity and pressure

Carbon and Energy Sources Photoautotrophs – use CO2 & energy from sunlight Chemoautotrophs – use CO2 & energy from organic cpds. Photoheterotrophs – use organic cpds. & energy from sunlight Chemoheterotrophs – use carbon and energy from same source (sugars, lipids, proteins, etc.)

Oxygen Requirements Aerobes (obligate aerobes) – need O2. Anaerobes – w/o O2 1. obligate anaerobes – can’t tolerate O2 2. Facultative anaerobes – live with or without O2, can ferment; ex. E. coli 3. Aerotolerant anaerobes – does not use O2, O2 does not harm them; ex. lactobacilli Microaerophiles – require low level of O2; ex. Helecobacter pylori

Other Chemical Requirements Nitrogen - Growth limiting nutrient, from organic (proteins, amino acids, DNA) and inorganic (metabolic waste); some bacteria can reduce nitrogen gas into ammonia (nitrogen fixation) Trace elements – selenium, zinc, copper, magnesium, iron, manganese, phosphorus Growth factors - vitamins, cofactors, coenzymes, essential amino acids; required by fastidious organisms

Temperature Requirements Optimum temperature Psychrophiles (-8˚C- 18˚C), Psychrotrophs (0˚C – 32˚C; food spoilers) Mesophiles (20-40˚C; human pathogens) Thermophiles (>45˚C; hot springs & compost piles) Hyperthermophiles (>80˚C; hot vents)

pH, Atmospheric and Osmotic Pressure pH - neutrophiles (optimum pH 6.5 – 7.5; human pathogens) - acidophiles (pH <4) - alkalinophiles (pH >8) Atmospheric pressure – 760 mmHg Osmotic pressure ( mOsm) - halophiles (salt loving); obligate halophiles (need high salt); facultative halophiles (don’t require but can tolerate salty conditions)

Culturing Microorganisms Clinical specimen – feces, saliva, CSF, urine, blood, skin, mucous membrane, discharge or diseases tissue. Culture – microbes that grow from an inoculum Obtaining pure culture ( progenitor, CFU) - aseptic technique

Pure Culture: Streak Plate Method Spread an inoculum (0.1 ml. on top) using sterile inoculating loop across the surface of solid medium in petri dish; incubate

Pure Culture: Pour Plate Method CFUs separated from each other using a serial dilutions. Final dilutions are mixed w/ warm agar in petri dish

Culture Media Nutrient broth, agar = complex polysaccharide 6 variety of culture media: 1.Defined – exact composition is known 2.Complex – contain variety of growth factors and can support a wider variety of microbes; TSA, nutrient broth 3.Differential – differentiates between organisms; blood agar, levine EMB agar (lac+ =purple; lac- =pink) BLOOD AGAR (BAP) Strep. Pyogenes – beta hemolysis (clear yellowish zone, complete) Strep. Pneumoniae – alpha hemolysis (greenish halo, partial) Enterococcus – gamma hemolysis (no red cells hemolyzed, no change around medium) E. Coli fermented sugar, produced acid (turn red phenol to yellow) and produced gas (bubble)

cont. variety of culture media 4. Selective – contain substance that either favor or inhibit microbial growth; sabouraud dextrose agar (slightly low pH is selective for fungi); Mc Conkey agar selective for gm. - and lactose fermenting McConkey agar = selective for gm. – E. coli and differentiates lactose Fermenting E. coli (red to pink colonies) Nutrient agar Sabouraud dextrose agar

cont. variety of culture media 5. Reducing (anaerobic) – conducive to anaerobes, contain sodium thioglycollate, that combine with free O2 and remove it. 6. Transport – to carry clinical specimens a) gas-pak anaerobic system b) anaerobic glove box

Microbial Growth Logarithmic (exponential) growth – binary fission, greater yields than simple addition Generation time – time required for a pop. of cells to double in number Semilog graph arithmetic graph

Phases of Microbial Growth Curve Lag phase – cells adjusting to environment, synthesize enzymes Log phase- rapid growth & reproduction, increases logarithmically Stationary phase- nutrients are depleted and waste accumulates, rate of reproduction decreases; number of dying cells = number of cells being produced Death phase- cells die at faster rate than number of new cells produced

Measuring Microbial Growth Direct Methods a. viable plate counts - make serial dilutions; count # colonies on spread or pour plate from each dilution b. membrane filtration - large sample poured thru filter to trap cells, transferred to solid media and colonies counted c. microscopic count - sample placed on cell counter and viewed thru microscope d. electronic counter- device that counts cells as they interrupt an electrical current e. most probable number (MPN) – statistical estimate; the more bacteria in a sample, the more dilutions are required to reduce their # to zero Viable plate count

Measuring Microbial Growth Indirect method a) turbidity - use of spectrophotometer; a more turbid broth culture will have a greater population b) metabolic activity - measure nutrients, wastes c) dry weight – oragnisms are filtered from culture medium, dried, and weighed

Homework 1.Define terms: autotrophs, heterotrophs, aerobes, anaerobes, facultative anaerobes, thermophiles, psychrotrophs, halophiles, inoculum, culture, generation time, and logarithmic curve. 2.Describe blood agar as differential culture for gamma, beta and alpha hemolysis of organism. 3.What culture medium is selective for gram – and lactose fermenting E. coli? 4.Describe measuring bacterial growth by viable plate count and turbidity methods. 5.Discuss at least 3 physical and chemical requirements for microbial growth in the laboratory. 6.Discuss the phases of microbial growth curve. 7.List clinical specimens used in doing microbial culture.