Transcript RCC Lab 7 S14 mod

Lab #7
Microbial growth and metabolism
• So far what we know:
• Colony morphology and cell morphology (rod vs cocci)
• Motility
• Oxygen requirement
• Gram stain, endospore, and acid fast reaction
• This information not enough to identify a particular
organism
• In order to identify an organism specifically (species)
biochemical and metabolic tests need to be performed
• Microorganisms are very diverse in their ability to:
• 1) Ferment/ utilize particular substrates as food
• 2) Produce specific enzymes that allow them to metabolize
Microbial growth and metabolism
• Metabolism = sum of processes occurring within the cell
that lead to the production of energy (ATP)
• Requires enzymes – proteins that carry out the chemical
processes within the cell
• Anabolism – synthesis of new bonds and larger molecules
(requires energy)
• Catabolism – breakdown of bonds and macromolecules (releases
energy)
• Anabolism and catabolism occurs via redox reactions
• Overview of metabolism:
Enzymes
Substrate
Product(s)
Microbial growth and metabolism
• Major nutritional needs:
• Energy source
• Carbon source
• Nitrogen source
• Phosphorous, sulfur
• Water (hydrogen, oxygen)
• Vitamins
• Minerals
How do microorganisms
get nutrients in their natural
habitat?
• Environment
How do microorganisms
get nutrients in the lab?
• Growth media
• Physical needs:
• Temperature
• Oxygen
• Osmotic pressure (salt concentration)
• pH
Growth Media – Pg. 59-61
• Classified based on composition (ingredients) and
function
• Solid vs. Liquid
• Solid media contains Agar
• Synthetic vs. Non-synthetic
• Synthetic = artificial; exact chemical composition (formula)
is known
• Non-synthetic = contains natural (unpurified) mixtures;
exact chemical composition is not known (digested proteins,
beef, peptone, animal/plant extract)
Growth Media – Pg. 59-61
• General purpose vs. Minimal
• General = allows many different organisms to grow
• Minimal = contains very specific nutrients  will allow
specific organisms to grow
• Enriched – contains complex ingredients (blood, yeast,
serum, brain infusion)
• Reducing – chemicals added to remove oxygen (thio,
cysteine)
• Buffered – chemicals added to stabilize pH of media (two
phosphate salts – KH2PO4 and K2HPO4)
Growth Media
• Selective vs. differential
• Selective = allows some organisms to grow but prevents
others (salt, pH, dyes, and toxins)
• Differential = different species will grow but will look different
(sugars, pH indicators)
Mannitol Salt Agar:
• Selective for
Staphylococcus species
• Differential for S. aureus 
able to ferment sugar
mannitol (yellow color)
Growth Media
• Media must be sterilized before use
• Autoclave
• Steam at high pressure
• 121oC and 15 psi for 15 minutes  achieves sterilization
• Radiation
• Filtration – heat liable
chemicals (antibiotics, dyes)
Biochemical Testing – Pg. 64-66
• How do you know a reaction has occurred?
• 1) Patterns and location of growth
• Aerobic vs. anaerobic
• 2) Turbidity (cloudiness)
• Increase  growth
• Decrease  hydrolysis/ breakdown substance in media
• 3) Physical condition of the medium
• Liquefaction  hydrolysis/
breakdown of substances in media
• Loss of fluidity (coagulation)
 liquid media becomes solid
• Gas production  bubbles can be detected
Biochemical Testing
• How do you know a reaction has occurred?
• 4) Colorimetric changes
• A result of pH change  detected by pH indicator
• Non-pH changes  a reagent is used to detect the
production of a particular substance
• Table on pg. 65
pH Indicators
Memorize pH indicators and color reactions!
pH
Indicator
Very
acidic
Acidic
Neutral
Basic
Phenol red
-
pH <6.8 =
yellow
pH 6.9 - 8.0 =
red
pH >8.0 =
magenta/ hot
pink
Litmus
-
pink
purple
blue
Bromocresol
Purple
-
yellow
burgundy
violet
Methyl red
pH <4.4 =
red
pH 5 - 6 =
orange
pH >6.3 =
yellow
-
Biochemical Testing
• Things to keep in mind for biochemical tests (pg. 66):
• Controls
• Must include a control for comparison & to validate the test
• Everything will be the same except one factor (m/o)
• A positive result will look different from the control
• Proper recording of results
• Positive result
• Reaction has occurred (color change, gas production, turbidity,
etc.)
• Indicate using a + sign
• Negative result
• No reaction has occurred (looks the same as control)
• indicate using a - sign
Sugar Fermentation – Pg. 71-74
• Bacteria have the ability to breakdown certain
carbohydrates to produce energy (ATP)  Fermentation
(pathway)
• Carbohydrates = sugars (made up of C, H, and O)
• Fermentation does not require oxygen (anaerobic)
• Fermentation of sugars results in the production of:
• Acid or an alcohol
• Gas (CO2) – in some cases
• Overview of fermentation:
Sugar  Pyruvic acid  acids/alcohol + ATP
(maybe CO2 gas)
Sugar Fermentation
• Fermentation can be detected using a media that includes
a sugar and a pH indicator
• Positive sugar fermentation reaction:
• A = acid production (yellow color change)
• A/G = acid production (yellow) and gas in Durham tube
• SA = slow reaction (orange color change)
• Negative sugar fermentation reaction:
• B = base production (pink/magenta color change)
• (-) = No change (looks exactly like
the control)
Sugar Fermentation Experiment - DEMO
• Types of sugars:
• Glucose
• Lactose
• Mannitol
• Sucrose
• Record results on pg. 74
Water Analysis Project and Lab Report
• Pg. 131 – 138
• Test for presence of coliform (E. coli)  G- rod bacteria
found in the GI tract
• Perform three tests (Pg. 136):
• Presumptive
• Confirmed
• Completed
• Today start the presumptive test
• Obtain 3 water samples A, B, and C  inoculate 1mL of each
sample into a separate lactose broth tube
• Inoculate 1mL of the (+) and (-) controls into separate Lactose
broth tubes
• Make sure all 5 tubes are properly labeled  Incubate the tubes
• Read results in 24-48 hrs (Wed – Fri open lab hours)
Water Analysis Project and Lab Report
• Presumptive test results (table on pg. 133)
• Positive: Acid and gas production = “a/g”
(color change to yellow and gas bubble in Durham tube)
• On Wed-Fri open lab: Use positive tubes to streak an EMB
(Eosin Methylene Blue) plate  Confirmed test
• One plate for each positive result!
• Confirmed test results
• Positive: Metallic green colonies
• Obtain a single colony from the positive streak plate and inoculate it
into a lactose broth  Completed test
• Completed test results
• Positive: Acid and gas produced
• Indicates the presence of coliform bacteria  fecal contamination!
Today’s Inoculations
• Nitrate reduction test (#15)  use loop to inoculate 3 Nitrate
broths:
• 1) B. subtilis
• 2) Alcaligenes faecalis
• 3) E. coli
• 4) Control tube  no inoculation
• Production of decarboxylase (#17)
• Use loop to inoculate Lysine broths with:
• 1) Enetrobacter aerogenes
• 2) Enterobacter cloacea
• 3) Klebsiella pneumoniae
• 4) Morganella morganii
• 5) Control tube – no inoculation
(total 12 tubes + 1 control)
Add 1mL of sterile
mineral oil on top of
EACH broth culture
AFTER inoculation
• Repeat using Arginine broths and Ornithine broths
Today’s Inoculations
• SIM Reations (#19)  1 tube, 3 tests
• Inoculate 4 SIM deeps using a needle:
• 1) Proteus vulgaris
• 2) E. coli
• 3) Citrobacter freundii
• 4) Morganella morganii
• 5) Control  no inoculation
• MR-VP Reactions (#20)  1 tube, 2 tests
• Inoculate 3 MR-VP broths using a loop:
• 1) Enterobacter aerogenes
• 2) E. coli
• 3) K. pneumoniae
• 4) Control  no inoculation
Today’s Inoculations
• Make sure each tube is properly labeled!!
• Name of media
• Inoculated bacteria
• Your name
• Date
• Place all your tubes in your basket and fasten them
with a rubber band
• Place basket in the incubator