Microbial Nutrition & Growth Microbial growth & significance ▪ Definition of microbial growth: an increase in number of cells rather than an increase in size ▪ Understanding the requirements for microbial growth ▪ Allow us to culture them and determine how to control their growth ▪ Specifically, of those microbes that cause food spoilage and disease Requirements for growth ▪ To grow and multiply, microbes need to synthesize cell components ▪ nucleic acids, proteins, lipid membranes, or cell walls ▪ To accomplish this task, microbes need sources of carbon, energy, and electrons ▪ Depending on the carbon and energy sources microbes are grouped into four basic groups ▪ The microbes studied in this course typically belong to the fourth group and we will focus on them!! Chemical Requirements for growth ▪ Several core chemicals are required to synthesize nucleic acids, proteins, lipid membranes, or cell walls ▪ This means that cell must make nucleotides, amino acids, phospholipids, and sugars built from major elements or macronutrients Trace elements and growth factors ▪ Trace elements or micronutrients are minerals essential for the function of certain enzymes ▪ Include ▪ copper ▪ zinc ▪ manganese ▪ molybdenum Oxygen Requirements ▪ Capnophiles are microbes that require higher concentration of carbon dioxide (up to ~10%) in addition to low oxygen levels Why do certain microbes need SOD and catalase? ▪ Aerobic metabolism involves the formation of reactive oxygen species (ROS) such as O2-/H2O2whichdamage cell structures, and certain bacterial species can eliminate their toxic effect using enzymes ▪ROS include singlet oxygen and hydroxyl radical -1O2/OH., the latter resulting from ionizing radiation ▪ Microbes use SODand/or catalaseas protective mechanisms ▪ SODconverts O2-to O2and H2O2 ▪ Catalase converts H2O2to O2and H2O The catalase test ▪ The catalase test is used in the laboratory to differentiate certain bacterial species such as ▪ Staphylococcus (facultative anaerobe, catalase positive) from ▪ Streptococcus (aerotolerant anaerobe, catalase negative) McGraw Hill - Photographic Atlas – B. Chess ▪ Phagocytic cells use toxic forms of oxygen to kill ingested pathogens ▪ Hydrogen peroxide can be used as an antimicrobial agent though the catalase activity limits its application Physical requirements for growth - Temperature ▪ Categories of microbes based on the temperature ranges for growth Psychrotrophs are also called psychrotolerants and include the pathogen Listeria monocytogenes ▪ Typically, human pathogens are mesophiles ▪ (Optimum growth temperature is ~ the body temperature) Effect of temperature ▪ Minimum growth temperature - microbes are able to conduct metabolism ▪ Maximum growth temperature – microbes continue to metabolize ▪ Optimum growth temperature – highest growth rate ▪ Growth rate plotted against temperature ▪ Growth of Escherichia coli on nutrient agar at three different temperature Temperature and bacterial growth Chancroid Variable temperature requirements of certain pathogens ▪ Treponemapallidum (the causative agent of syphilis) prefers lower temperatures ▪ Lesions are first seen on exterior parts of the body including lips, tongue, and genitalia Effect of pH on microbial growth ▪ Neutrophiles ▪ Optimum pH near neutral ▪ Acidophiles ▪ Grow best in acidic habitats ▪ Alkalinophiles ▪ Live in alkaline soils and water ▪ Most pathogens are neutrophiles, grow best in the range 6.5-7.5 - the pH range of most tissues and organs in the human body ▪ Helicobacter pylori (associated with gastric ulcers) is not an acidophile but an acid-tolerant (produces urease) ▪ Vibrio cholerae, the causative agent of cholera, can thrive at a pH as high as 9.0. Effect of Osmotic Pressure ▪ Osmotic pressure is the pressure exerted on bacterial cells by their environment Isotonic Hypertonic (shriveling of the cytoplasm) ▪ Hypotonic: the bacterial cell gains water and swells to the limit of its cell wall ▪ Obligate halophiles - adapted to grow under high osmotic pressure, require high salt concentration ▪ Facultative halophiles - tolerate high salt concentration ▪ Staphylococcus species that colonizes the surface of the skin How do we culture microbes? ▪ Microbes in natural environments ▪ Live in mixed communities, relationships may be antagonistic, synergistic, or symbiotic ▪ Form biofilms (complex structures and regulated interactions) ▪ Biofilms impact or benefit humans ▪ Clinical significance - disease ▪ Environmental significance bioremediation/waste water treatment Microbes in the laboratory are often cultured as pure cultures ▪ To cultivate (or culture) microbes ▪ A sample (inoculum) is placed into/on sterile broths or sterile agar media Slants ▪ Aseptic techniques are used ▪ Microbes that grow from an inoculum are called a culture Deeps ▪ Cultures visible on solid media as discrete units are called colonies (CFUs) Petri plate Characteristics of bacterial colonies can help in the process of identification * * * * * Mixed culture * * Pure culture Serratia marcescens ▪ Clinical specimens must be properly collected ▪ Placed in sterile containers ▪ Labeled ▪ Promptly transported to a clinical laboratory to ▪ Avoid death of the pathogen ▪Minimize growth of members of the normal microbiota ▪ Transport media are often used to move specimens from one location to another ▪ If clinical specimens are not handled or cultured properly ▪ Pathogenic bacteria may be missed or may not survive ▪ Leading to wrong diagnoses!!!! Microbes in clinical specimens -Clinical laboratory Health care professionals collect specimens according to the CDC - Standard precautions ▪ Sterile swabs ▪ Needle aspiration ▪ Intubation ▪ Catheter ▪ Clean catch method ▪ Sputum (coughing/catheter) ▪ Biopsy ▪ Clinical specimens are collected to identify a suspected pathogen ▪ Specimens often include microorganisms of thenormal microbiota ▪ Suspected pathogen in the clinical specimen must be isolatedfrom the normal microbiota in culture ▪ Several techniques can be used to isolate the pathogen in pure culture (axenic culture) Handling and culturing clinical specimens in the laboratory Techniques to isolate the suspected bacterial pathogen in pure culture or axenic culture ▪ Quadrant streaking technique of isolation ▪ The technique enables only a qualitative analysis to separate the species in the specimen or detect the colonies of the pathogen in clinical specimens ▪ Subculturing to achieve a pure culture Techniques to isolate the suspected bacterial pathogen in pure culture or axenic culture ▪ The method of serial dilutions involves dilution of the specimen ▪ Each dilution is then plated using the pour-plate or the spread-plate techniques of isolation ▪ This method enables quantitative analysis: count the number of cells in the specimen from the CFUs ▪ Plating serial dilutions of the specimen ▪ Pour plate method ▪ Spread plate method What criteria must be met to successfully culture a specimen? All nutrients requiredby the microbe in the specimen, including energy source and growth factors for fastidious pathogens Sufficient moisture(water) Appropriateoxygenlevels ▪ Sterilization of media and aseptic techniques are designed to minimize contamination of the specimen Properly adjusted pH of the medium Suitable osmoticpressure Proper temperatureof incubation for growth Synthetic versus complex media ▪ Nutrient broth (NB) is the liquid version of the medium without agar ▪ TSB: Trypticase soy broth ▪ TSA:Trypticase soy agar Synthetic & complex media Selective and differential media help establish the presence of pathogens ▪ A selective medium contains ingredients that inhibit the growth of some organisms while encouraging the growth of others Sabouraud dextrose agar selects for the growth of fungi while inhibiting the growth of bacteria Nutrient agar - pH 7.3 Sabouraud agar - pH 5.6 Blood agar ▪ Blood agar plate (BAP) is a complex differential medium, which is typically used to grow fastidious species and detect hemolytic activity Alpha-hemolysis Beta-hemolysis No hemolysis (gamma-hemolysis) ▪ Streptococcus pyogenes ▪ Streptococcus pneumoniae ▪ Staphylococcus epidermidis MacConkey ▪ MacConkeyis aselective and differential medium for detection and differentiation of Gram negative bacilli (enteric bacilli) Colonies of Klebsiella on MacConkey BAP and b-hemolysis by Staphylococcus aureus Pictures: Di Bonaventura (Bio 265 lab) EMB – Eosin methylene blue ▪ EMBisaselective and differential medium for detection of Gram negative bacilli that ferment lactose (Coliforms) Eosin methylene blue agar E. coli (fecal coliform) forms dark purple with a metallic green sheen colonies Other coliforms (general coliforms) form dark purple colonies ▪ Eosin and methylene blue inhibit the growth of Gram positives ▪ EMB allows for differentiation of E.coli from the general coliforms such as Klebsiella or Enterobacter ▪ Gramnegative bacilli that do not ferment lactose form colorless colonies MSA–Mannitol salt agar ▪ MSAisaselective and differential medium for detection and differentiation of Gram positive cocci – Staphylococcus species ▪ High salt concentration (7.5%) to select for Staphylococcus species while inhibiting the growth of other species ▪ Fermentation of mannitol in MSA helps differentiate Staphylococcus species MSA is a selective/differential medium Picture: Di Bonaventura (Bio 265 Lab) ▪ MSA is used for isolation and detection of Staphylococcus aureus Clinical implications of bacterial growth and culture media Enriched medium ▪ Bacteria can be “fastidious” in a laboratory setting, Nesseria gonorrhoeae or Haemophilus influenzae ▪ Some cannot be grown on culture media: Mycobacterium leprae (armadillos) or Treponema pallidum (rabbits) ▪ Some others are obligate intracellular parasites (chlamydias and richettsias) and require cultures of living cells ▪ Chocolate agar used to culture H. influenzae and N. gonorrhoeae MacConkey BAP Sabouraud Differential Selective EMB Selective and differential Not selective or differential (complex medium) MSA Chocolate agar medium Anaerobic microbial cultures, media, and systems ▪ Obligate aerobes or facultative anaerobes can be grown in a standard incubator, but obligate anaerobes would not grow in presence of oxygen ▪ Stab cultures ▪ Reducing media Containing reducing agents (thioglycolate) that remove oxygen and indicators (resazurin) ▪ Enzymatic systems (Oxyrase) ▪ Anaerobic culture system (anaerobic GasPakTM jar) An Anaerobic Chamber ▪ To culture anaerobes that cannot tolerate exposure to oxygen Preserving Bacterial Cultures ▪ Bacterial cultures are stored by decreasing cell’s metabolism ▪ Prevent exhaustion of all nutrients and excessive accumulation of waste products ▪ Storage for short period of time ▪ Refrigeration (weeks to months) ▪ Long-term storage ▪ Deep-freezing (years) ▪ Lyophilization (freeze-drying) (decades) ▪ Involves removing water from a frozen culture using an intense vacuum. Lyophilized cultures are restored by adding them to liquid media Bacterial growth by binary fission – asexual reproduction ▪ Generation time is the time required for a bacterial cell to grow and divide into two cells ▪ Or - Generation time is the time it takes for a population of cells to double in number Bacterial growth by binary fission – asexual reproduction ▪ Under optimal conditions, E. coli or S. aureus cells have a generation time of ~ 20 min ▪ To calculate the total number of cells in a population, we multiply the original number of cells by 2n ▪ Nt= N0 x 2n ▪ Nt = number of cells in a population at a given time ▪ N0 = initial number of cells in the population ▪ n= number of generations (division) Typical microbial growth curve ▪ When bacterial cells are grown in a closed system (batch cultures), nutrients are depleted, and waste products accumulate in the medium ▪ The bacterial growth curve exhibits distinct phases ▪ The generation time is calculated during the log phase because cells multiply at a constant rate ▪ In later stages of log phase endospore-forming-bacteria would initiate the process of sporulation How do we measure microbial growth? ▪Working with clinical specimens can involve quantitative analysis such as assessing a significant bacteriuriain urine samples Direct Methods Indirect Methods Direct microscopic count Turbidity* Electronic counters: Coulter counter & Flow cytometry Metabolic activity Plate counts* Dry weight Filtration* MPN Direct method Microscopic cell count ▪ Microscopic count using the Petroff-Hausser counting chamber or a Hemocytometer ▪ The counting chamber of the special glass slide contains a counting grid ▪ The cell suspension is loaded on to the counting chamber covered by a coverslip ▪ Counts the total number of cells, or dead/live cells in a specimen Direct Method: Viable Plate Counts ▪ Serial (stepwise) dilutions of the specimen - the dilution factor at each step is constant ▪ Dilutions are plated ▪ After incubation, colonies (CFUs) on plates are counted ▪ 25-30 to 250-300 colonies/plate or CFUs (colony-forming units) – countable plate!! Direct Method - Membrane Filtration ▪ Bacterial cells are filtered out from the sample and retained on the surface of the filter ▪ The filter is then transferred on the surface of a culture medium ▪ Colonies form from the bacterial cells on the surface of the filter. The number of cells in the specimen is calculated from the CFUs Direct Method: Counting Bacteria by Membrane Filtration ▪ Bacteria are filtered out and retained on the surface of the filter ▪ The filter is transferred to a culture medium ▪ Colonies arise from the bacterial cells on the surface of the filter Indirect Method Turbidity ▪ This method uses an instrument called a spectrophotometer ▪ The amount of light hitting the detector is inversely proportional to the number of bacteria ▪ The less light is transmitted, the more bacteria in the sample ▪ (including both dead & live cells – biomass