MICROBIAL GROWTH DR HALA ALDAGHISTANI Microbial Growth Microbial

MICROBIAL GROWTH DR. HALA AL-DAGHISTANI

Microbial Growth Microbial growth: Increase in cell number, not cell size! Physical Requirements for Growth: § Minimum growth temperature § Optimum growth temperature § Maximum growth temperature Temperature Five groups based on optimum growth temperature 1. Psychrophiles 2. Psychrotrophs 3. Mesophiles 4. Thermophiles 5. Hyperthermophiles

Physical Requirements for Growth: p. H and Osmotic Pressure Most bacteria grow best between p. H 6. 5 and 7. 5: Neutrophils Some bacteria are very tolerant of acidity or thrive in it: Acidophiles (preferred p. H range 1 to 5) Molds and yeasts grow best between p. H 5 and 6 Hypertonic environments (increased salt or sugar) cause plasmolysis Obligate halophiles vs. facultative halophiles Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

WATER ACTIVITY QUANTIFIES WATER AVAILABILITY IN AN ENVIRONMENT; DECREASES WITH INCREASING SOLUTE CONCENTRATION. PLASMOLYSIS: HYPERTONIC SOLUTIONS; CYTOPLASM WATER LOSS; COMPATIBLE SOLUTES. OSMOTOLERANT: GROWS OVER A WIDE RANGE OF WATER ACTIVITY; FUNGI > BACTERIA. HALOPHILE: “SALT-LOVING”; REQUIRES > 0. 2 M SODIUM CHLORIDE.

Chemical Requirements for Growth: Carbon, N, S, P, etc. § Carbon § Half of dry weight § heterotrophs use organic carbon sources § Autotrophs use inorganic carbon sources § Nitrogen, Sulfur, Phosphorus § N, S Needed for protein sythesis § N, P used for NA and ATP synthesis § S in thiamine and biotin § Phosphate ions (PO 43–) § Also needed K, Mg, Ca, trace elements (as cofactors), and organic growth factors Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Nutrient Concentration Effects in Batch Cultures • Total growth will increase until limiting nutrients are exhausted (included oxygen for aerobes) or metabolic by products accumulate that change environmental conditions to inhibit growth (toxicity). • Growth rate will also increase with increasing nutrient concentration up to a some maximum value, beyond which there is no effect (transporters are saturated with there substrate.

Oxygen Requirement Types 2 to 10% atm O 2 Super Oxide Dismutase (SOD): Superoxide radicals (O 2 • -), or superoxide anions (OH 2¯². Superoxide radicals go to hydrogen peroxide & O 2. Catalase: hydrogen peroxide go to water & O 2.

Toxic Forms of Oxygen § Singlet oxygen: O 2 boosted to a higher-energy state § Superoxide free radicals: O 2– § Peroxide anion: O 22– § Hydroxyl radical (OH ) Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Biofilms Fig 6. 5 Microbial communities form slime or hydrogels Starts via attachment of planctonic bacterium to surface structure. Bacteria communicate by chemicals via quorum sensing Sheltered from harmful factors (disinfectants etc. ) Cause of most nosocomial infections Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

ADVANTAGES OF BIOFILMS 1. CELL-TO-CELL CHEMICAL COMMUNICATION 2. QUORUM SENSING, ALLOWS BACTERIA TO COORDINATE THEIR ACTIVITY AND GROUP TOGETHER INTO COMMUNITIES THAT PROVIDE BENEFITS NOT UNLIKE THOSE OF MULTICELLULAR ORGANISMS. 3. WITHIN A BIOFILM COMMUNITY, THE BACTERIA ARE ABLE TO SHARE NUTRIENTS AND ARE SHELTERED FROM HARMFUL FACTORS IN THE ENVIRONMENT, SUCH AS DESICCATION, ANTIBIOTICS, AND THE BODY'S IMMUNE SYSTEM. 4. FACILITATING THE TRANSFER OF GENETIC INFORMATION'S (CONJUGATION). Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Culture Media § Culture medium: Nutrients prepared for microbial growth. Have to be sterile (not contain living microbes) § Inoculum: Microbes introduced into medium § Culture: Microbes growing in/on culture medium § Chemically defined media: Exact chemical composition is known (for research purposes only) § Complex media: Extracts and digests of yeasts, meat, or plants, e. g. : § Nutrient broth § Nutrient agar § Blood agar Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Agar § Complex polysaccharide § Used as solidifying agent for culture media in Petri plates, and slants § Generally not metabolized by microbes § Liquefies at 100°C § Solidifies ~40°C Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Anaerobic Culture Methods § Use reducing media, containing chemicals (e. g. thioglycollate) that combine with O 2 § Are heated shortly before use to drive off O 2 § Use anaerobic jar § Novel method in clinical labs: Add oxyrase to growth media Oxy. Plate (no need for anaerobic jar) Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Capnophiles: Aerobic Bacteria Requiring High CO 2 § Low oxygen, high CO 2 conditions resemble those found in § intestinal tract § respiratory tract and § other body tissues where pathogens grow § E. g: Campylobacter jejuni § Use candle jar, CO 2 generator packets, or CO 2 incubators Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings 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; Mac. Conkey agar, EMB, mannitol salt agar etc. Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Enrichment Media/Culture § Encourages growth of desired microbe § Example: Assume soil sample contains a few phenoldegrading 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 Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

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 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! Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Streak Plate Method 3 or 4 quadrant methods

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. Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

The Growth of Bacterial Cultures Binary fission – exponential growth Budding Generation time – time required for cell to divide (also known as doubling time) Ranges from 20 min (E. coli) to > 24 h (M. tuberculosis) Consider reproductive potential of E. coli Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Fig 6. 13 Figure 6. 12 b

Bacterial Growth Curve Phases of growth § Lag phase § Exponential or logarithmic (log) phase § Stationary phase § Death phase (decline phase) Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

THE LAG PHASE - THIS PERIOD OF LITTLE OR NO CELL DIVISION. DURING THIS TIME, HOWEVER, THE CELLS ARE NOT DORMANT. THE MICROBIAL POPULATION IS UNDERGOING A PERIOD OF INTENSE METABOLIC ACTIVITY INVOLVING, SYNTHESIS OF ENZYMES AND VARIOUS MOLECULES THE LOG PHASE (EXPONENTIAL PHASE) - THE CELLS BEGIN TO DIVIDE AND ENTER A PERIOD OF GROWTH, OR LOGARITHMIC INCREASE. CELLULAR REPRODUCTION IS MOST ACTIVE DURING THIS PERIOD, THE STATIONARY PHASE - THE GROWTH RATE SLOWS, THE NUMBER OF MICROBIAL DEATHS BALANCES THE NUMBER OF NEW CELLS, AND THE POPULATION STABILIZES. (PERIOD OF EQUILIBRIUM) Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

WHAT CAUSES EXPONENTIAL GROWTH TO STOP IS NOT ALWAYS CLEAR. 1. THE EXHAUSTION OF NUTRIENTS 2. THE ACCUMULATION OF WASTE PRODUCTS, AND HARMFUL CHANGES IN PH MAY ALL PLAY A ROLE. THE DEATH PHASE - THE NUMBER OF DEATHS EVENTUALLY EXCEEDS THE NUMBER OF NEW CELLS FORMED, AND THE POPULATION ENTERS THE DEATH PHASE, OR LOGARITHMIC DECLINE PHASE. Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Direct Measurements of Microbial Growth Viable cell counts: Plate counts: Serial dilutions put on

Figure 6. 15, step 1

Additional Direct Measurements Direct microscopic count: Counting chambers (slides) for microscope A specially designed slide called a petroff-hausser cell counter is used in direct microscopic counts. -motile bacteria are difficult to count by this method -Dead cells are bout as likely to be counted as live ones. Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

FILTRATION Filtration is the method of choice for low counts M. O. § Membrane filters for fluids. Pore size for bacteria: 0. 2 – 0. 4 m Pore size for viruses: 0. 01 m At least 100 ml of water are passed through a thin membrane filter whose pores are too small to allow bacteria to pass . Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Estimating Bacterial Numbers by Indirect Methods Spectrophotometry to measure turbidity OD is function of

THE MOST PROBABLE NUMBER (MPN) METHOD - ANOTHER METHOD FOR DETERMINING THE NUMBER OF BACTERIA IN A SAMPLE IS THE MOST PROBABLE NUMBER (MPN) METHOD - THIS STATISTICAL ESTIMATING TECHNIQUE IS BASED ON THE FACT THAT THE GREATER THE NUMBER OF BACTERIA IN A SAMPLE, THE MORE DILUTION IS NEEDED TO REDUCE THE DENSITY TO THE POINT AT WHICH NO BACTERIA ARE LEFT TO GROW IN THE TUBES IN A DILUTION SERIES. - IT IS USEFUL WHEN THE GROWTH OF BACTERIA IN A LIQUID DIFFERENTIAL MEDIUM IS USED TO IDENTIFY THE MICROBES (SUCH AS COLIFORM BACTERIA, WHICH SELECTIVELY FERMENT LACTOSE TO ACID, IN WATER TESTING). Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

METABOLIC ACTIVITY - INDIRECT WAY TO ESTIMATE BACTERIAL NUMBERS IS TO MEASURE A POPULATION'S METABOLIC ACTIVITY ( ACID OR CO 2, IS IN DIRECT PROPORTION TO THE NUMBER OF BACTERIA PRESENT). DRY WEIGHT -FOR FILAMENTOUS BACTERIA AND MOLDS, THE FUNGUS IS REMOVED FROM THE GROWTH MEDIUM, FILTERED, AND DRIED IN A DESICCATOR. IT IS THEN WEIGHED Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings

Measuring Microbial Growth - Overview Direct Methods Indirect Methods § Plate counts § Turbidity § MPN § Metabolic activity § Direct microscopic § Dry weight count § Filtration Copyright © 2006 Pearson Education, Inc. , publishing as Benjamin Cummings