Announcements Instagram account for the lab: vuubio201 Lab 3 handout-take 1 before you leave class DO THE PRE-LAB and READ THE ENTIRE DOCUMENT BEFORE you take the quiz Quiz due at midnight Wednesday 2/5 As you are doing the lab, READ DIRECTIONS CAREFULLY Late lab notebook 5 points off Lab notebook due Monday 2/10

Chapter 4: Dynamics of Prokaryotic Growth, Part 2

What are the environmental factors that influence microbial growth? Did you know that there are also nutritional requirements also for microbial growth? 2 Minute Brainstorm and Recap

Why do bacteria need specific nutrients? What are the four major macromolecules of life? Carbohydrates Proteins Lipids Nucleic acids Monosaccharide Amino acid Fatty acids Nucleotides What are their monomers? Bacteria require the major elements to make these macromolecules!

Required Elements That Make Up Bacterial Cell Parts C carbon O oxygen H hydrogen N nitrogen S sulfur P phosphorus K potassium Mg magnesium Ca calcium Fe iron

Amino Acids Nucleic Acids Sugars Lipids Amino Acids: Cysteine

Enzymes:

Oops…Out of Stock! Have you ever been baking cookies and realized you have only half the amount of a certain ingredient? That specific ingredient (let’s say chocolate chips) is the limiting nutrient The number of cookies you make DEPENDS on the amount of chocolate chips Bacteria have limiting nutrients also Fe iron P phosphorus Determines the maximum level of microbial growth possible- All bacteria need it, but there is not enough to go around.

Too Much of A Limiting Nutrient=Explosive Growth! Problems with pollution in water (extra phosphorus) allows for algae to grow out of control! How do you control the growth then? Remove Phosphorus

What is the source of carbon for bacteria? Heterotroph – Hetero = different – troph = nourishment Uses organic carbon Autotroph – Auto = self Use inorganic carbon from CO 2 What is the energy source for bacteria? Phototroph – Photo = sunlight – Extract energy from the sun Chemotroph – Chemo = chemical – Extract energy from chemical compounds

Where do these different bacteria get their energy and carbon from? Type Energy SourceCarbon source Photoautotroph Photoheterotroph Chemolithoautotroph Chemoorganoheterotroph (most common) lith = Stone- Inorganic organo= organic Organic compounds Organic compounds CO 2

To learn how bacteria can be used for our benefit or treated with drugs in infections, we need to be able to grow and study them in the lab…

Recreating Bacteria Growth Requirements in the Lab 2 types of media: – Liquid media- water-based broth containing nutrients (example: LB broth) – Solid media- broth with agar added that turns into a gelatinous solid when cooled to room temperature (example: agar plates)

Why Use Agar? Polysaccharide extracted from marine algae Microbes can not degrade it Not destroyed at high temperatures – Can be autoclaved to sterilize Solidifies below 45 o C – Add in heat sensitive nutrients Once solidified, remains that way until heated to 95 o C – Covers the temperature range of microbial growth

We Have An Agar Plate with Bacteria Pictured Below…Now What? Colony- a distinct mass of cells -contains at least one million bacterial cells to be visible -colony started from one single bacterium (clonal)

No longer dividing Still dividing/expanding

How do I study only the blue colonies?

Colony selection Aseptic technique

Streaking for isolation

Isolated colonies Using an isolated colony, you can start a pure culture -a population descended from one single cell (clonal) -contains only one species

Growing Bacteria in the Lab: Most Can Eat A Good Meal Like Us! Complex media- variety of ingredients in varying amounts (you never know the exact composition), “tasty soup” Example- Nutrient agar Grows most types of bacteria General lab use; easy to make Example- Blood agar Grows most types of bacteria General lab use

Growing Bacteria in the Lab: But, Some Need Specific Meals! Chemically defined media- uses exact amounts of pure chemicals Used in experiments where nutrition is strictly controlled or for “picky” bacteria Example: Neisseria gonorrhoeae – Require very specific nutrients to grow (46 total!)

Comparison Between Complex and Chemically Defined Media Nutrient broth Peptone Meat extract Water Glucose salts broth Glucose Dipotassium phosphate Monopotassium phosophate Magnesium sulfate Ammonium sulfate Calcium chloride Iron sulfate Water Complex Chemically Defined

Activity: Selective Media We are a mixed population of bacteria: stand up If you have a red paper: eat peanuts for your diet to grow If you have a blue paper: eating peanuts will inhibit your growth We feed both bacteria crushed up peanuts with agar on the same plate: if you grow stay standing. If you don’t, sit down. Who lives? This is a selective media RED

Selective Media Selective media- inhibits the growth of certain species, while allowing the growth of others MacConkey agar is selective, because only Gram negative bacteria can grow on it (inhibits Gram positive bacteria from growing)

Activity: Differential Media We are a mixed population of bacteria: Stand up If you have a red paper: you produce a protein which kills red blood cells and turns them white If you have a blue paper: you produce a protein which partially kills red blood cells and turns them green We feed both bacteria red blood cells with agar (blood agar) on the same plate: does everyone grow? How can you tell the difference between the bacteria? This is a differential media AND REALLY HAPPENS with Bacteria! Partial lysis of red blood cells: Green Full lysis of red blood cells: White YES

Differential media- does not inhibit microbes, but it allows you to differentiate species with color changes MacConkey agar is both selective and differential; it inhibits growth of Gram positives, and indicates which Gram negatives can ferment lactose with a color change Differential Media

Question Hektoen enteric agar- -contains inhibitors to prevent Gram positive growth -If the bacteria produce H 2 S, the colony appears black -If the bacteria can ferment the sugars in the media, it turns yellow/orange Is it differential? Selective? Both?

How Do Bacteria Grow In Nature? BIOFILMS Bacteria that attach to a surface and live as a community encased in polymer “slime”

Biofilm Protected! Secrete extracellular polymeric substances (EPS) Planktonic bacteria (motile) Biofiolm bacteria (immobile)

Bad Biofilms Dental plaque that leads to tooth decay Contaminate/damage surfaces of industrial machinery Infections from biofilms on catheters

Good Biofilms