Microbial GrowthChapter 6

ObjectivesDescribe some techniques used to grow microorganismsExplain the requirements for the growth of microorganismsExplain the different phases of bacteria growth

Techniques on Bacterial GrowthSeveral steps involved:Obtain a sample for analysisFollow the five Is to purify and characterize the organismInoculateIncubateIsolateInspectIdentify

First things firstObtain a sample for analysisAny object can serve as source of microorganismsSkin, blood, water, food etc

1. InoculationPlace sterile mediumSterile loopMedium with nutrient to support growthLiquidSolidLive organism

Give examples of microbes grown in living organisms?

Viruses, bacteria(E.coli, mycobacterium leprae)

2. IncubationProper growth environmentTemperatureGas

3. IsolationResult of inoculation and incubationFurther isolation by subculturing to obtain pure cultures

4. InspectionObserve macroscopic and microscopic charactersColonies shapes, size, colorStain characteristics

5. IdentificationSeveral approachesMorphological characteristics: Cell wall, cell shape etcAlso useful for identifying eukaryotesDifferential staining: Gram staining, acid-fast staining etcBiochemical tests: Determines presence of bacterial enzymes

Bacteria Growth RequirementsBacteria have both Chemical requirementsCarbon, oxygen, nitrogen, sulfur, phosphorous and trace elementsPhysical requirementsTemperature, pH, osmotic pressureOthers

Chemical requirements1. Carbon(to make structural molecules)Autotrophs use CO2 as carbon source and inorganic salts as N source (plants, and some bacteria)Heterotrophs use organic carbon sources (animals and most bacteria)Carbon compounds (source of energy)

Chemical requirementsNitrogenUsed to make amino acids and proteinsWhat is the source of NSulfurUsed in amino acids, thiamine and biotinSource: sulfate SO42 or H2SPhosphorusUsed in DNA, RNA, ATP, and membranesSource: Inorganic phosphate (PO43)

5. OxygenOxygen requirements can divide microorganisms into 5 Groups Need OxygenPreferOxygenIgnoreoxygenOxygen Is toxicRoughly2-10%oxygen

Table 6.1 The Effect of Oxygen on the Growth of Various Types of Bacteria

Q&AOxygen in the atmosphere is essential for human life. How can some bacteria grow in the absence of oxygen?

Oxygen easily reduced to toxic productsSuperoxide radical Hydrogen peroxideHydroxyl radicalAerobes produce protective enzymesBasis of Different Oxygen Sensitivities

Aerobic Organisms (aerobes)Require molecular oxygen (O2) Obtain energy by respirationProduce SOD and CatalaseObligate (strict) aerobe (absolute need for O2)Examples Micrococcus and Corynebacterium (skin)Pseudomonas aeruginosa (burn infections)Bordetella pertussis (whooping cough)Mycobacterium tuberculosis (lung-disease tuberculosis)

Facultative AnaerobesGrow in presence or absence of oxygen (Grows better in presence of oxygen) Produce SOD and catalaseRespiration (in oxygen) and fermentation (absence of oxygen)Examples:Escherichia coli, Salmonella, and Shigella Staphylococcus (e.g. S. aureus)Saccharomyces (S. cereviceae) - bakers yeast

Anaerobic Organisms (Anaerobes)Obligate (strict) anaerobeDo not produce both SOD and catalase Grown in strongly-reducing media (thioglycolate) or in an anaerobe jarExamples:Clostridium (tetanus, gas gangrene, and botulism)Bacteroides (normal intestinal flora)Associated with appendicitis

Figure 6.6 & 6.7Anaerobic JarWhen water is mixed with the chemical packet, H2 and CO2 are generatedCatalyst combines H2 and O2 to form waterMethylene blue turns colorless when the O2 is removed

Aerotolerant AnaerobesSimilar to anaerobes (do not require O2)Grow in oxygen Produces SOD and some produce catalaseGrow better in a candle jar than in airExamples:Lactobacillus and StreptococcusSugars lactic acid (lactic acid bacteria).Lactobacillus - lactic acid rodsImportant in agriculture/food industryStreptococcus - lactic acid cocci Streptococcus pyogenes- sore throat, tonsillitis, scarlet feverStreptococcus pneumoniae - bacterial pneumonia

MicroaerophilesRequire higher CO2 and lower O2 concentrations than that in airProduce SOD and catalase Grow best in candle jar (why?)Examples Branhamella catarrhalis non-pathogenic (mouth)Neisseria spp (pathogenic/non-pathogenic species).Neisseria gonorrhoeae - (STD- gonorrhea) Neisseria meningitidis - (meningitis)Campylobacter jejuni - food poisoning and gastroenteritis Campylobacter fetus abortion sheep/cattle

SOD and Catalase

GroupSuperoxide dismutaseCatalaseObligate aerobes and most facultative anaerobes (e.g. Escherichia)++++++Most aerotolerant anaerobes (e.g. Streptococci)+++--Obligate anaerobes (e.g. Clostridia, Methanogens, Bacteroides)----Microerophiles(e.g. Neisseria)+++-- (some ++)

Physical RequirementsTemperaturepHOsmotic PressureOthers: radiation, atmospheric pressure and chemical require

6. TemperatureEach m/o has a minimum, optimum, and maximum growth temperature (Cardinal temperatures)Note the names of organisms

Temperature Optima

OrganismMinimumOptimumMaximumPsychrophiles(cold-loving) (fridge- listeria)-10oC15oC20oCPsychrotrophs(skin, micrococcus)0oC25oC30oCMesophilesModerate temp15oC20-40oC45oCThermophiles(Heat-loving) 45oC55-70oC80oCExtreme thermophiles(extremophiles) (volcanic vents)65oC85-95oC105oC

Figure 6.2Food Preservation TemperaturesPsychrophiles mostly found in deep sea, polar regions; seldom cause problems in food preservationPsychrotrophs more common than psychrophiles; cause spoilage in foodMesophiles most common type of microbes; include most of the common spoilage & disease m/osThermophiles cannot grow at temp below 40oC; commercial sterilization cannot kill the endospores but not consider a public health problemHyperthermophiles grow at 121oC near deep-sea hydrothermal vents

7. pHType or organisms base on growth optimum pH:AcidophilesOptimum: pH 0 - pH 5.5NeutrophilesOptimum: pH 5.5 - pH 8AlkalophilesOptimum: pH 8.5 - pH 11.5

Bacteria and viruses can survive low stomach pH to get to intestinesHelicobacter pylori lives in stomach under mucus layerHow can it survive in such low pH?

8. Osmotic PressureHypertonic environments, (High salt or sugar conc.), cause plasmolysis (water loss and cytoplasm shrinkage)Key principal in food preservationHalophiles:Grow optimally at > 0.2MNon-halophiles< 0.2 M NaCl concentrationFacultative halophilesNormally grow best w/o salt but Can grow in salt presence Extreme (obligate) halophiles: require >2M (30% salt)

BiofilmsMicrobial communitiesForm slime or hydrogelsBacteria attracted by chemicals via quorum sensingShare nutrientsSheltered from harmful factors

Pseudomonas aeruginosa biofilm

Growth of BacteriaSome terms:Culture medium: Nutrients prepared for microbial growthSterile: No living microbesInoculum: Microbes introduced into the mediumCulture: Microbes growing in/on culture medium

Culture MediaCan be:Liquid (Broth)Semi-solid (Deeps)Solid (plate)BrothPlateSlantDeep

AgarComplex polysaccharide Used as solidifying agent for culture media in Petri plates, slants, and deepsGenerally not metabolized by microbesLiquefies at 100CSolidifies ~40C

Chemical Content of MediaChemically defined (synthetic) All components known and quantified e.g.Minimum mediaComplex/non-syntheticSome components not known or quantified e.g.Nutrient agarNutrient broth

Tables 6.2, 6.4

Functional Types of MediaEnrichedContain nutrients that favor growth of select bacteria e.g. blood agar, chocolate agarDifferentialDistinguishes bacteria growing on same plate e.g. blood agarSelectiveEncourage growth of particular bacteria while suppressing the growth of unwanted bacteria e.g. MacConkey agar

MacConkey agar is both selective and differentialSelect for G(-), Differentiate- lactose fermenters

Preparing Pure CulturesSeveral techniques used to prepare pure culturesStreak platePure culture:Contain only one species or strainColonyArising from single cell or sporeColony is often called a colony-forming unit (CFU)

Streak PlateFigure 6.10ab

Special Culture TechniquesContainment levels (Biosafety level- BSL) BSL1 (low individual and community risk) m/os that handle in a basic microbiology teaching labNon-pathogenic E. coliBSL2 (moderate individual risk, low community risk) m/os that present a moderate risk of infection; operators should wear gloves, lab coats, etcC. difficile, MRSA, Salmonella, BSL3 (high individual risk, low community risk) m/os are highly infectious airborne pathogens; the lab should be negatively pressurized and equipped with air filters to prevent release of pathogens.Mycobacterium tuberculosis, Bacillus anthracis, West Nile virus, SARS coronavirus, Salmonella typhi.BSL4 - (high individual risk, high community risk)Ebola virus, Lassa virus, smallpox

E.Coli inside body grows best at body temp (37)Skin swab grows best at room temperature as it grows on skin surface

**Nitrogen is needed for proteinsPhosphorus is needed for phospholipid bilayer, ATP, DNA, RNATrace elements- cobalt, molybdenum, only needed in very small amounts*Nitrogen- some take preformed n2 from plants while others acquire from environment**Tube consists of medium raging from varying levels of O2 content from 20% on the top and 0% in the bottom. Different electron acceptors can be used such as sulfur

Or fermentation will be used*Antioxidants scavenge for these radicals to remove from body as they can be very harmfulSuperoxide dismutase(SOD) converts Superoxide radical into H2O2Catalase converts H2O2 to H2O and O2

A simple lab test is to add m/os to the radical and if oxygen is produced, it is a catalase positive m/os

*Micrococcus- found in skin as always found in the presence of oxygen

*E.coli- very versatile in Large intestine, but also outside the bodyRespiration-using oxygen to carry out cellular processes and produce energy for the cellFermentation- mostly glycolysis, m/os produce lactic acid, or alcohol***What else causes pneumonia- viruses, mycoplasma*Microaerophiles require about 5%CO2 *atmospheric pressure- bottom of the ocean***121o temp at which microbiological sterilization occurs**Alkaline pH- oceans as it is slightly basic. Especially salt pans, where bicarbonate exists)

Bacteria burrows into the mucus, and creates a microenvironment producing ammonia causing damage to the cell. This is a possible cause to stomach ulcers.*Hyper- higherBacteria biofilms a problem in hospitals, fro example, within the cathoders, become a source of infection (one might break off). They are also sheltered and antibiotics may not be effective.***Differential- Beta completed lysis the blood cells, Alpha partially lysed the blood cells, Gamma did not lyse the blood cellsDiluting the concentration of your culturesSeparating the individual cells*