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

Ccarbon

6

12.011

Ooxygen

8

15.999

Hhydrogen

1

1.008

Nnitrogen

7

14.007

Ssulfur

16

32.065

Pphosphorus

15

30.974

Kpotassium

19

39.098

Mgmagnesium

12

24.305

Cacalcium

20

40.078

Feiron26

55.845

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

Feiron26

55.845

Pphosphorus

15

30.974

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 CO2

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?

TypeEnergy Source Carbon source

Photoautotroph

Photoheterotroph

Chemolithoautotroph

Chemoorganoheterotroph(most common)

lith = Stone-

Inorganic

organo= organic

Organiccompounds

Organiccompounds

CO2

CO2

CO2

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 45oC– Add in heat sensitive nutrients

• Once solidified, remains that way until heated to 95oC– 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

Streaking for isolation

Streaking for isolation

Streaking for isolation

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 agarGrows most types of bacteriaGeneral lab use; easy to make

Example- Blood agarGrows most types of bacteriaGeneral 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 mediaRED

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

White vs. Green colors

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

QuestionHektoen enteric agar--contains inhibitors to prevent Gram positive growth-If the bacteria produce H2S, 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”

BiofilmProtected!

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

machineryInfections from

biofilms on catheters

Good Biofilms