Microbiology - Chapter 7 & 8

Microbiology - Chapter 7 & 8

Microbial Growth – Bacteria reproduce by “binary fission”, a cell divides into two, two to four, four to eight, etc.

Cell division can occur quite rapidly depending on nutrient levels, temperature, etc. E. coli can divide every 20 minutes (time to double – generation time) in nutrient media at 37 degrees C. The numbers get so large we express them as the log of the number of cells.


Microbial Growth is affected by two major factors:

Environmental: temperature, pH, Osmotic conditions

Chemical: Proper concentrations of C, H, O,

N, P, S, some trace elements, and some organic cofactors


Bacterial Growth Curve:

A= Lag phase

B= Log phase

C= Stationary phase

D= Death Phase

Know this and be able to explain what is occurring during each phase.

Bacteria that produce endospores may not have a death phase. Why?


Why study such a growth curve?

Helps us understand how microbes grow under different conditions

Helps us understand how pathogen grow in our body

Helps us study the effect of different chemicals, osmotic conditions, even the effect of temperature on bacterial growth.

Ex. What would the growth curve for E.coli look like if we incubated at 4 degrees Celsius? At 65?


How do we measure growth of bacteria in a growth curve?

Direct cell count using stained slides that have a grid for counting. (Tedious, a real pain)

Indirect: Serial dilution, plates are innocculated - incubated and colonies counted. Number of colonies X dilution factor gives the number of bacteria.

Microbiology. Chapter 7 -8

How do we measure growth of bacteria in a growth curve? Direct cell count. Tedious and time consuming





Measure cloudiness in a test tube as the number of cells increase (turbidity) using a spectrophotometer. Correlate this data with a standard plate count and now just use the turbidity measurement – look up number from a chart from then on. Saves time and money.



Coulter counter. Electronically counts number of bacteria as bacteria pass through a tiny tube. Expensive.


Physical factors that affect bacterial growth;

Mesophiles : grow best moderate temp. 25 – 40 degrees most of our lab microbes

Psychrophiles: adapted to survive and grow at cooler temp., even in the frig (below 25 degrees)

Listeria (in cheeses and meat)

Thermophiles: adapted to and grow at much higher temp.

Thermus aquaticus, from oceanic vents, survives at

60 degrees C

Leprosy bacilli prefer 30 degrees, most pathogens prefer 37 degrees.


Physical/Chemical factors that affect bacterial growth; pH: measure acidity and alkalinity of media

Bacteria grow best at pH range of 6.5 to 7.5

Fungi grow better at slightly acid condition (5.0 to 5.5)

Sabaraud dextrose and Potato dextrose agars

One pathogen, Helicobacter pylori , is adapted to and survives in stomach acid (cause of ulcers)

Hydrostatic pressure: some bacteria grow really well deep in the ocean at pressures that crush submarines like and “egg”


Physical/Chemical factors that affect bacterial growth; pH: measure acidity and alkalinity of media

Osmotic pressure; relative salt concentrations in water solutions

Hypertonic: higher salt concentrations, slows or stops bacterial growth; salt preservative in meat some prefer higher salt: Halophiles some survive and thrive, Vibrio bacteria, V. cholera

Hypotonic: lower salt, fresh water, net flow water into cells, bacteria have rigid cell wall resist rupture

Isotonic: equal solute (salt) no net flow, preferable


Chemical factors that affect bacterial growth: Nutrition

How microbe acquire nutrients. C, H, O, N, S, P, Ca, Mg, etc

Carbon:

Autotroph: producers, photosynthetic, use CO

2 and

H

2

O, sunlight as energy, make their own food

Heterotroph: require preformed food, digestive and absorptive, most microbes

Chemoautotroph: unique metabolism, use chemical energy from inorganic molecules, Sulfur and

Iron


Chemical factors that affect bacterial growth: Nutrition

How microbe acquire nutrients. C, H, O, N, S, P, Ca, Mg, etc

Oxygen:

Obligate aerobes : require molecular oxygen (as final electron acceptor in catabolism)

Pseudomonas spp.

Obligate anaerobes : require atmosphere with no O

2 an organic molecule is final electron acceptor in catabolism (like a fermentation pathway)

Clostrida grow in “Brewer Jar”

Facultative anaerobes : grow with or without O

2

, usually are also fermenters, like E. coli

Microaerophile : grow best in lower oxygen and higher carbon dioxide, Strep., candle jar


Problems with oxygen: oxygen can be toxic, it oxidizes and destroys vital cell chemicals; aerobic organisms have enzymes and systems to handle it

SOD: superoxide dismutase, enzyme that chemically alters toxic oxygen free radicals and toxic high energy “singlet oxygen” to less toxic hydrogen peroxide

Catalase: Converts hydrogen peroxide to oxygen and water


Nitrogen: Found in all the amino acids, nitrogenous bases of nucleic acids, etc.

Hydrogen: found in all biological molecules, Carbs, fats, proteins, nucleic acids, etc

Phosphorous: found in nucleic acids, ATP, and phospholipdids of membranes

Sulfur: found in 2 or 3 amino acids of microbes

Trace elements: inorganic elements needed in very tiny concentrations (manganese, cobalt, Zn, Cr)


Organic cofactors:

Vitamins

Required by certain bacteria, “fastidious” hard to grow

Coenzymes Many microbes produce their own from scratch, source of our supplements (one a day, GNC)

Fastidious organisms may require enriched media to get them to grow

(blood, eggs, etc)

Some organisms are almost impossible to culture because of their strict parasitic-fastidious nature (syphilis, leprosy)

Microbiology chapters 7 - 8 part 2

Metabolism

Catabolism

Anabolism Both occur simultaneously in cells

Catabolism eventually produces the chemical energy (ATP) required for all cellular functions such as anabolism (synthesis), membrane transport, etc.


ATP – Adenosine triphosphate, universal cellular energy

Cyclically made and energy is stored and then broken down and the energy is released


Note: ATP is a ribonucleotide, it has ribose, a nitogenous base (adenine), and phosphate. The high energy bond of the terminal of the three phosphates is the one cyclically broken and regenerated.

Sugars like glucose can be broken down in a catabolic pathway controlled by many cellular enzymes. Some of the energy released by the breaking of covalent bonds is harvested and stored in the “energy” bonds of ATP.

Most any biomolecule can be used for energy; we will focus on the “catabolism” of glucose (a monosaccharide) and later show how the others are involved (lipids, AA, etc)


Quick review on enzymes

Organic catalyst (made of carbon, speed up rate of chemical reactions)

Made of protein; chains of Amino acids in a specific sequence that fold and coil into specific shapes. Their shape is key to understanding their function. (remember shape determines function) Also, shape is easily affected by changes in temperature. So, heat or cold can cause enzymes to slow down or even stop.

An enzyme lowers “activation energy” – energy required for a reaction to begin


Quick review on enzymes

Substrates are the material that are acted on by the enzyme

Enzymes are often named using the name of the substrate and adding “ase”. Sucrase breaks down sucrose to glucose and galactose. Enzyme driven reactions are often reversible.


Aerobic metabolism; specifically glucose catabolism

This stuff is hard “Just do It”

Goal:

1. List the three stages of glucose catabolism

2. Know the basic steps of each stage

3. Know how much ATP is made at each stage per molecule of glucose

4. Starting products and end products, other important carriers (NAD)

5. The difference between substrate level phosphorylation and oxidative phosphorylation

6. Theory of chemiosmosis and ATP production at the membrane of the mitochondria


Glucose is a hexose, monosaccharide, C

6

H

12

O

6

It is systematically broken down through three related “pathways” to

Carbon dioxide (CO

2

) and Water (H

2

O)

Overall Formula:C

6

H

12

O

6

+ ___ O2  CO

2

+ ___ H

2

O

The three stages:

Glycolysis (anaerobic) (in cytoplasm)

Krebs cycle (aerobic)

Electron transport (with chemiosmosis) (aerobic)

(in mitochondria)


Glycolysis: Anaerobic, no oxygen required, linear NZ pathway

Begins with ______

End products _________

How much ATP? _______

How many carrier molecules? ____

Name the carrier molecule. ____

Where in the cell? _______

What happens after if the organism

Is an aerobe? _____

Facultative? ______

Strict anaerobe? ______

Aerobe deprived of oxygen? ____


Krebs cycle (TCA, Citric acid cycle) Aerobic stage, Occurs in the fluid of the Matrix


This is a cyclic “pathway”

Pyruvic acid is first acted on by an NZ and a coenzyme (COA). The end product is Acetyl-Coa and a CO

2 molecule.

Remember this occurs twice for each glucose molecule. (One glucose is split into two pyruvic acid molecules.)


The acetyl-COA reacts with an enzyme and another substrate

(a 4-C molecule called oxaloacetic acid) to produce Citric

Acid, a 6 carbon tri-carboxylic acid; 3 carboxyl groups

Several enzymes systematically oxidize the citric acid into a

5-C acid, then a 4-C acid and eventually back to the original oxaloacetic acid – thus a cycle. Each time the terminal carboxyl group is removed a CO

2 molecule is produced.

Thus, one glucose, causes the cycle to turn twice, each turn produces 3 CO cycle)

2

(one at Acetyl COA step and two in the

Now for the hard part. Understanding that an oxidation reduction reaction is going on at each step. (Here **Krebs**), at glycolysis, and even electron transport)

Let’s first review oxidation- reduction (aka: redox)


Oxidation – Reduction

Organic molecules like glucose have covalent bonds between C-C, C-H, C-O, O-H

C

6

H

12

O

6

When the molecule is broken down -

, the covalent bonds are broken

– electrons are removed and transferred to carrier molecules.

Oxidation is the removal of electrons and/or adding Oxygen

In Glycolysis the glucose is broken into two Pyruvates,

The electrons and a H + are transferred to a carrier, NAD.

NAD gains the electron (and Hydrogen too), it is reduced to NADH, thus oxidation and reduction go together .


Oxidation – Reduction

Look again at glycolysis.

Glucose is oxidized and the carrier NAD is reduced.

For every glucose, two Carriers are produced

2 NADH (what happens to them, they have to be regenerated – oxidized back to NAD)

Aerobes eventually produce CO

2 and H

2

O

Thus oxygen is the final electron acceptor( producing

Water).

Anaerobes use a different set of enzymes, a

Fermentative pathway that generates other acids, alcohols or gasses (lactic acid, ethanol, CO

2

)

** electron acceptor is an “organic molecule”**

If no regeneration of NAD, the glycolysis pathway shuts down and the organism dies – no ATP


Glycolysis, no oxygen, fermentation, only 2 ATP per molecule of glucose

Glycolysis, with oxygen, followed by Krebs and electron transport, can generate much more ATP (sometimes as much as 36). Aerobic mechanisms are much more energy efficient.

In the Krebs cycle many more carrier molecules like NADH are generated and thus lead to more ATP. (Other carriers FAD, NADP – we just use

NAD as a representative type of carrier).

The constantly turning of the cycle produces a steady stream of reduced carriers (NADH) which pass the electrons to a set of carrier-processor molecules imbedded in the membrane of the Mitochondria. These carriers are called the “electron” transport chain.


Return to Krebs and show how it works with electron transport chain.

Note how glycolysis and Krebs are working together. Note that each produces reduced carriers that need to be processed.


The electrons are passed down the chain and end up being added to oxygen. The Hydrogen ion (H+) is pumped out (proton pump) and flows back in at special sites to be added to the Oxygen and electron to form Water. Energy is conserved (harvested; stored) in the bonds of ATP


Theory of Chemiosmosis: Proton pump, increased H+ ion concentration, flow through ATP synthase related channel, energy is harvested in high energy bonds of ATP. Enough to generate 34 more ATP.


Fermentation: Many microbes ferment sugars and other substrates to make ATP without oxygen

See pg 234 in text: NADH reduces pyruvate and ethanol and carbon dioxide are produced

Other end products are seen: lactic acid, acetic, acid

We use biochemical tests and the end products of sugar fermentation to ID bacteria (charts in Bergey’s) ** later in lab, particularly with unknown 2

Some bacteria, like E. coli and Bacillus use nitrogen electron acceptors to regenerate NAD. Nitrate and Nitrite reduction are examples. Pg 233 in text. The enzyme system is called Nitrate reductase


Other fuels

Proteins: digested to amino acids

Amino acids are :

‘deaminated’ : amino group removed, the reulting ‘acid’ can be further metabolized, more ATP decarboxylated: carboxyl group removed, the end products then enter glycolysis or Krebs to make ATP


• Lipids are catabolized to Glyerol and Fatty acids

• Glycerol easily enters glycolysis and Krebs just like pyruvate

• Fatty acids are chopped into 2 or 3 acid fragments that are broken downt to carbondioxide

• Even nucleic acids – OH SO MUCH

MORE!!! Take biochem.

Microbiology - Chapter 7 & 8

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