Microbial Growth and Culture

Question 1

Explain this statement as completely as possible (in detail): Bacteria grow.

Answer 1

the cells are becoming more numerous, that the bacteria are reproducing. And so when we say growth, and we’re talking about micro organisms, we almost always are referring to their reproductive process. The reproductive potential of bacteria is staggering.

Question 2

Why is a log scale used for the y-axis of bacterial growth curves?

Answer 2

It gives us an easier way to see the line/shape of the growth graph.

It is easier to predict growth linearly compared to exponentially, due to the unchanging slope as time increases on the X-axis.

Question 3

In this course, unless otherwise stated, logarithms always have 10 as their base.

Solve the following without a calculator. log(10),

Answer 3

1

Question 4

log(1,000)

Answer 4

3

Question 5

log(10,000,000)

Answer 5

7

Question 6

log(1).

Answer 6

0

Question 7

Estimate log(4,000) without a calculator. Explain how you performed those estimations.

Answer 7

log(4,000) is between log(1000) and log(10,000) so it’s between 3 and 4 but closer to 3 so I’d say it’s equal to 3.

Question 8

Estimate log(30) without a calculator. Explain how you performed those estimations.

Answer 8

log(30) is between log(10) and log(100) so it’s between 1 and 2 but closer to 1 so I’d say it equals 1.

Question 9

Diagram a bacterial growth curve with 4 phases. Explain what is happening in each phase.

Answer 9

• Lag: cells are adapting to culture conditions
(synthesis of needed enzymes, IF ANY)
– Example: transferring organisms from an old culture
(dormant) to fresh media
– Example: transferring organisms from a fresh glucose
based broth to a lactose based broth
• Exponential (AKA ‘Log phase’): rapid growth; appears
linear on log scale graph
–Nutrients available; waste concentration is low
–Growth at the maximum rate possible for the conditions
• Stationary: nutrient levels decrease and waste
levels increase, causing stress; in response, growth
slows, death increases, and total # cells is constant
–Bacillus and Clostridium bacteria make endospores
• Death: lack of nutrients or toxic waste molecules increase
death rate

Question 10

Some growth curves have only 3 phases. Which phase is not always present in a bacterial growth curve?

Answer 10

The death phase is not always present in continuous culture where constant nutrients are supplied.

Question 11

E. coli were grown for 24 hours in a tube of broth containing glucose as the main energy source. A loop was used to transfer a small amount of the culture to 2 new tubes, one containing the same glucose broth and another containing a lactose-based broth. Diagram the growth curves for the cultures in the 2 new tubes.

Answer 11

The E. coli can ferment lactose and glucose. However, e.coli will consume glucose at continuous rate since it was already placed in glucose. However, the bacteria in lactose will not grow for a little while as they develop enzymes needed to break down lactose. Then the growth curve will eventually meet the other one.

Question 12

Yersinia enterocolitica are pathogenic bacteria. The doubling time of these bacteria is 25 minutes at 37 C and 300 minutes at 4 C. Diagram the growth curves for 2 Y. enterocolitica cultures–1 at each temperature.

Question 13

Write the equation for the exponential growth of bacteria. Explain what each term means.

Answer 13

Nt=No x 2^(t/d)

• Nt is total number of cells at time, t
• N0 is the number of cells to start with (at
time 0)
• t is the time that has passed
• d is the doubling time, the time it takes for
the bacteria to undergo binary fission
• t and d have the same units

Question 14

If a broth tube is inoculated with 3,000 cells which immediately enter exponential growth, how long will it take for there to be 1,500,000 cells, if the doubling time is 40 minutes?

Answer 14

Nt= 1500000 CFU
No= 3000
t= ?
d= 40

1,500,000/3,000 = 2^t/40 500 = 2^t/40
2^8 = 2^t/40
8 = t/40
t=320 min

Question 15

If a broth tube is inoculated with 1,000 cells which immediately enter exponential growth, how many cells will there be after 6 hours if the doubling time is 45 minutes?

Answer 15

Nt=No x 2^(t/d)

N(0) = 1,000
N(t) = ?
T = 6 hours x 60 min/hr = 360 min
D = 45 min

N(t) = 1,000 x 2^(360/45) = 1,000x2^8 = 256,000

Question 16

If a broth tube is inoculated with 3,600 cells which immediately enter exponential growth, and there are 230,000 cells after 3 hours, what is the doubling time?

Answer 16

?

Question 17

A broth tube is inoculated at 9 AM. At 2 PM there are 600,000 cells. At 3 PM there are 4,800,000 cells. How many cells were put in the tube?

Answer 17

?

Question 18

Sanitizer

Answer 18

• Sanitizer: reduces number of microbes to a safe level; some are specified to be safe for food contact surfaces

Question 19

disinfectant

Answer 19

• Disinfectant: applied to inanimate objects to
reduce microbial load (the number of microbe
cells/virus particles)

Question 20

antiseptic

Answer 20

• Antiseptic: applied to human tissues to reduce

microbial load

Question 21

antimicrobial

Answer 21

• Antimicrobial: in some contexts refers to
disinfectants and antiseptics, in other contexts
refers to antimicrobial medications such as
antibiotics

Question 22

sterilant

Answer 22

• Sterilant: destroys all microbes including bacterial

endospores

Question 23

bactericidal

Answer 23

• Bactericidal: destroys vegetative bacterial cells

Question 24

bacteriostatic

Answer 24

• Bacteriostatic: prevents vegetative bacterial cells from reproducing

Question 25

virucidal

Answer 25

• Virucide: inactivates virus particles so they cannot infect; also spelled viricide

Question 26

sporicidal

Answer 26

• Sporicidal: see sterilant; destroys all microbes including bacterial endospores

Question 27

fungistatic

Answer 27

Fungistatic means it doesn’t kill the fungi, it just stops them from reproducing right. Cidal is to kill or inactivate Static is to prevent from reproducing.

Question 28

degerming agent

Answer 28

• Degerming: removing microbes from a surface

Question 29

Cleaner

Answer 29

• Cleaner: detergent/soap that removes dirt, therefore

reducing the number of microbes by removing them

Question 30

What is a phenolic? Explain how phenolics work.

Answer 30

Phenolic compounds used as antiseptics or disinfectants include pure phenol and substitution products with halogens and alkyl groups. They act to denature and coagulate proteins and are general protoplasmic poisons. Phenol (carbolic acid) is one of the oldest antiseptic agents. It goes after the plasma membrane.

Question 31

Explain hexachlorophene works.

Answer 31

Hexachlorophene is actually mostly used as an antiseptic applied to the skin, although it could be used as a disinfectant applied to inanimate objects. It goes after the plasma membrane.

Question 32

Explain how chlorhexidine works.

Answer 32

Chlorhexidine is used as an antiseptic on the skin. Sometimes patients are washed in a diluted solution of chlorhexidine. The brand name product that contains it is called Hibiclens. It goes after the plasma membrane.

Question 33

Name 2 halogen-based control agents. Explain how they work.

Answer 33

• Iodine
– Disrupts protein synthesis and membranes
– Oxidizes proteins
– Disinfectant
• Chlorine
– Bleach: hypochlorous acid (HOCl)
– Oxidizes DNA, proteins, membranes (everything)
– Potentially a sterilant, but mostly used to sanitize and
disinfect

Question 34

What is a quat ? Explain how quats work

Answer 34

These are like super soaps. They all have some structure similar to this. There’s a nitrogen atom connected to 4 organic carbon chains. That nitrogen atom has a positive charge, which makes it act like a super detergent, so powerful that it will denature proteins and completely disrupt the membranes of microorganisms.

Question 35

How do alcohol-based antiseptics and disinfectants work? Explain the relationship between water and alcohol-based antiseptic effectiveness.

Answer 35

• Ethanol (ethyl alcohol) and isopropanol
(isopropyl alcohol)
– Denature proteins, disrupt membranes
– Require water to be effective
– Disinfectant, antiseptic if contact maintained,
otherwise simply a degerming agent
(mechanical removal of dirt/microbes)

Question 36

What is the oligodynamic effect? To which chemicals does the term oligodynamic effect apply? Give 2 examples of antiseptics based upon the oligodynamic effect.

Answer 36

The oligodynamic effect refers to the very strong effect of a very small amount of silver ions. Other ions can do it, like mercury and copper, but silver is the one that’s most widely used now. you can denature the proteins of the microbes with incredibly small amounts of silver.

• Oligodynamic action
– Denature proteins at very low concentrations
– Silver nitrate eye drops are used to prevent
neonatal gonorrheal infections; erythromycin
(abx) now used in US
– Silver sulfadiazine antiseptic cream is applied
to burns
– Thimerosal is a mercury-based preservative
in adult vaccines)

Question 37

What is the purpose of adding nitrites to refrigerated meats such as hot dogs?

Answer 37

The purpose of adding nitrites to meats is to prevent the germination of bacterial endospores, which is potentially a problem. Nitrites specifically prevent bacterial endospores from turning back to vegetative cells.

Question 38

Name 3 organic acids used as preservatives. How do they work?

Answer 38

One type of preservative that’s commonly used as an organic acid, things like benzoic acid, sorbic acid or sometimes you’ll see calcium propionate, which is an acid derivative. They inhibit the metabolism of fungi and many bacteria which will then stop them from reproducing quickly on the food. Another type of preservative is nitrites. Nitrites specifically prevent bacterial endospores from turning back to vegetative cells.

• Organic acids
– Inhibit metabolism
– Sorbic acid, benzoic acid, and calcium propionate
– Control molds and bacteria in foods and cosmetics
• Nitrites prevents endospore germination
– Refrigerated meats, other foods

Question 39

How does ethylene oxide work? What is it used for?

Answer 39

One way that medical supplies can be sterilized: gas sterilization. The ethylene oxide is a chemically reactive gas that modifies the proteins, the lipids, the DNA on any organism that happens to be in that chamber. Will make these devices, these surgical instruments, sterile. You usually use this when heat sterilization is something you can’t do, such as with a plastic probe or something like that.

• Ethylene oxide
• Alkylates proteins, DNA, RNA
• Long exposure time needed
• Used on heat-sensitive medical instruments

Question 40

Rank the following microbes in terms of their resistance to disinfectants:
Gram-positive bacteria, Gram-negative bacteria, mycobacteria, bacterial endospores, vegetative protozoa, protozoan cysts, fungi, non-enveloped viruses, enveloped viruses.

Answer 40

Prion is a infectious misfolded protein. Prions are capable to withstand the effects of proteases, heat, ionising radiation and chemicals. The probable reason of the high endurance of prion are the presence of high degree of folds and formation of chain linked amyloid fibers.

Endospores are thick walled resistant structures formed by some gram positive bacteria during unfavourable conditions. Endospores can resist heat, radiation, desiccation,and enzymatic digestion. Outer coat of Endospores protect from chemical and enzymatic damage. Thick peptidoglycan layer below the coat protects from high temperature. Small acid soluble proteins tightly bind to the DNA and protects from radiation damage. A chemical known as dipicolinic acid comprises 10% of the spore’s dry weight and provide heat resistance.

Mycobacteria are rod shaped slow growing bacteria. Mycobacteria has hardiness due to the presence of mycolic acid in the cell wall and high GC content of the DNA.

Certain protozoa during unfavourable conditions form cyst. Cysts have thick walls containing chitin, acid fast lipids etc.

vegetative Protozoa are free living feeding form and have stability due to the presence of cell membrane and protinaceous pellicle in certain forms.

Gram negative bacteria acquires their stability due to the presence of outer membrane outside the cell wall.

Fungi and fungal spores are protected by the coat of chitin, various heat shock proteins and hydrophilins.

viruses without envelope are more stable due to the absence of sensitive outer coat.

Gram positive bacteria are less stable than gram negative bacteria due the absence of outer membrane.

Enveloped viruses are least stable due the presence of highly sensitive coat of lipid bilayer.

Question 41

What explains the different disinfectant resistances and the ranking of Gram-positive bacteria, Gram-negative bacteria, mycobacteria, and endospores?

Answer 41

The most disinfectant-resistant microbes are the endospores of bacteria. Bacterial endospores are the hardest to destroy. The reason is that they have no metabolism–they’re dormant. And their water has been replaced with an organic acid. And perhaps for some other reasons, the endospores of bacteria are the hardest things to kill. if you can destroy those, you’ve destroyed all microbes. The next hardest to kill are mycobacteria. Mycobacteria have a waxy outer layer and they reproduce very slowly. Those two things mean it’s very hard to kill mycobacteria. Their waxy layer probably makes it impossible for many disinfectants to get into the cell and damage the vital components. So it’s very hard to come up with something that kills Mycobacterium.

Question 42

What explains the different disinfectant resistances and the ranking of non-enveloped viruses and enveloped viruses?

Answer 42

We have fungi, viruses without envelopes, Gram positive bacteria, and viruses with lipid envelopes. And so the viruses with lipid envelopes are the most susceptible to any form of disinfection which is good for us. Some viruses have a protein coat with nucleic acids inside and some viruses have a protein coat with nucleic acids inside and an outside piece of lipid called an envelope. The envelope for in this lecture, you think of that envelope is being similar to a membrane.

Question 43

What explains the different disinfectant resistances and the ranking of vegetative protozoa and protozoan cysts?

Answer 43

of them as having a dormant form called a cyst, or a non dormant form. For example, the trophozoite form of Entamoeba histolytica. Trophozoites are vegetative protozoa. They reproduce, they are active. Cysts are inactive. They’re dormant. And it’s not surprising that the dormant form which is evolved to withstand long periods in a hostile environment, the dominant form resists disinfectants more than the vegetative form.

Question 44

Name the 3 cellular structures in microbe cells that are most frequently targeted by the disinfectants and antiseptics discussed in class. Do the disinfectants and antiseptics discussed in class have the potential of attacking those same targets in human cells? If so, how do we keep ourselves safe when using disinfectants? How do we keep ourselves safe when using antiseptics?

Answer 44

Cell wall/plasma membrane, metabolism, denaturing microbe proteins

Proteins, plasma membranes, and DNA or RNA are the most frequently target structures within a cell that is targeted by disinfectants and antiseptics. They do have the potential to target those same structures in human cells depending on their strength. We use protective/safety barriers depending on how strong a disinfectant is like gloves, masks, and goggles.
Ex: clorox wipe - has quats so it can kill microbes, but there is a dilution effect.

Question 45

If a broth tube is inoculated with 1,500 cells which immediately enter exponential growth, how long will it take for there to be 3,000,000 cells, if the doubling time is 30 minutes?

Question 46

If a broth tube is inoculated with 100 cells which immediately enter exponential growth, how many cells will there be after 8 hours if the doubling time is 30 minutes?

Question 47

If a broth tube is inoculated with 2,000 cells which immediately enter exponential growth, and there are 1,000,000 cells after 6 hours, what is the doubling time?

Question 48

A broth tube is inoculated at 9 AM. At 12 PM there are 6,400 cells. At 2 PM there are 1,000,000 400,000 cells. How many cells were put in the tube?