Microbiology Lecture 2 Review: Chapter 4,5 Dynamics and Control Microbial Growth

Lecture Review - By: Amma Genfi

Chapter 4 - Dynamics of Microbial Growth

1.    [04-01] Assume that only 100 cells of Escherichia coli are present in your potato salad. Explain how they can cause food poisoning if the salad is kept at room temperature for 5 hours?  – Lecture 2, Slides 2-3 –

E. coli has a generation time of 20mins. Therefore, within a period of 5 hours, 15 generation times will pass. Using the equation N_b = (Nº)(2^{(# of generation times)}) → Nb = (100cells)(2^15) = 3276800 ~ 3.27*10⁶ cells

2.    [04-02] Describe the phases of bacterial culture growth – Lecture 2, Slide 7 –

Lag phase – Bacteria lagging in growth because still adapting to environment.

Log phase – Period of exponential growth. Population doubles with each generation time.

·      Early – Production of primary metabolites; during this phase bacteria are the most sensitive to antibiotics b/c they are in process of synthesis of proteins, nucleic acid, cell wall

·      Late – Production of secondary metabolites; Used to help them compete with other bacteria for dwindling nutrients.

Stationary Phase – Population remains stable as rate of cell growth = rate of cell death. Nutrients are exhausted in media, instead nutrients from dead cell are used. Some gram-positive bacteria produce endospores during this phase.

Death Phase – Population rapidly declines as rate of cell death exceeds rate of cell growth. Population shrinks by large amount >90%.

Prolonged decline – Once population declined by 99% remaining live cells enter prolonged decline that is marked by gradual decrease in population. May last for many months.

3.    [04-03] Why are psychrotrophs and mesophiles important to human health? – Lecture 2, Slide 8 –

Psychotrophs have a growth range from 0ºC to + 30ºC with optimal growth temperature around +20ºC to +28ºC. → Microbes abundant around humans, can grow at +4ºC causing food spoilage in refrigerators.

Mesophiles have a growth range from +15ºC to 42ºC with optimal growth temperature around +30ºC to 37ºC. → Associated with birds, animals and humans, normal flora + pathogens.

4.    [04-04] Compare obligate aerobes, facultative anaerobes, obligate anaerobes, microaerophiles, aerotolerant anaerobes. Name human pathogens for each bacterial group – Lecture 2, Slide 9 –

Oligate aerobes: Grow only in the presence of oxygen b/c extract energy via aerobic cellular respiration only (Ex: Mycobacterium tuberculosis).

Facultative anaerobes: Grow best in aerobic conditions, but able to grow (slower) in anaerobic conditions with anaerobic respiration or fermentation (Ex: Escherichia coli).

Obligate anaerobes: Grow only in absence of oxygen and extract energy with anaerobic cellular respiration. (Ex: Clostridium tetani or botulinum)

Microaerophiles: Can tolerate low concentration of oxygen only (2-10%) (Ex: Helicobacter pylori)

Aerotolerant anaerobes: Indifferent to presence of oxygen because they do not have electron transport chain. Extract energy via fermentation (Ex: Streptococcus pyogenes)

5.    [04-05] Why should aerobic bacteria produce superoxide dismutase and catalase? – Lecture 2, Slide 10 –

To neutralize toxic forms of oxygen (superoxide radical and peroxide) formed during aerobic cellular respiration, they produce superoxide dismutase and catalase.

6.    [04-06] Explain the differences between obligate and facultative halophiles. Name bacteria that are facultative halophiles – Lecture 2, Slide 10 –  Halophiles are bacteria or archaea that can grow in the presence of high salt concentration. They produce small organic molecules that are effectively increasing the tonicity inside the cell enabling them to survive in hypertonic environment. Obligate halophiles can tolerate up to 20% of salt concentration. They are constantly producing these molecules and, therefore, can live in environments with high salt concentrations. Facultative halophiles can tolerate up to 10% of salt concentration and can regulate tonicity inside their cells by switching on and off the production of small organic molecules. Therefore, they can live in environments with normal and high salt concentrations. Examples of facultative halophiles: Bacillus subtilis (free-living organism), Staphylococcus epidermidis (human normal flora), Staphylococcus aureus (human opportunistic pathogen). Examples of bacteria sensitive to salt: Escherichia coli (human normal flora), Streptococcus pyogenes (human pathogen).

7.    [04-07] Give example of selective medium and explain how it works. Give example of differential medium and explain how it works – Lecture 2, Slides 13-14 – Selective medium contains selective agent that allows one group of microbes to grow while growth of others is inhibited. Thayer-Martin agar contains mixture of antibiotics inhibiting Gram-positive bacteria (vancomycin), Gram-negative bacteria (colistin and trimethoprim) and fungi (nystatin). The only microbes that can grow on the medium are species of genus Neisseria. MacConkey agar contains bile salts that inhibit the growth of gram-positive bacteria. Only Gram-negative bacteria can grow. EMB agar contains eosin and methylene blue that inhibit the growth of Gram-positive bacteria. Only Gram-negative bacteria can grow on this medium. Differential medium contains some components that allow differentiate growing bacteria into different groups. Differential medium can also contain selective agents and, therefore, be selective. MacConkey agar selects against Gram-positive bacteria. It also differentiates bacteria into fermenters and non-fermenters of lactose. If lactose present in medium is fermented by growing bacterium, the produced lactic acid changes the color of neutral red, the pH indicator, to pink. The growing lactose non-fermenter does not change the color of the medium. EMB agar selects against Gram-positive bacteria and differentiate lactose fermenters from non-fermenters. If lactose is fermented, produced lactic acid cause precipitation of eosin and methylene blue that stains the colony of lactose fermenters dark. In addition, the colonies of E. coli, a strong fermenter, also acquire distinctive green metallic sheen. Bloor agar is non-selective medium but allows differentiate growing bacteria into three groups: 1) alpha-hemolytic bacteria lyse red blood cell partially and form greenish halos around their colonies. They are moderate opportunistic pathogens. Example: Streptococcus pneumoniae; some strains of Staphylococcus aureus. 2) Beta-hemolytic bacteria lyse red blood cells completely and form clear halos around their colonies. They are highly aggressive pathogens. Examples: Streptococcus pyogenes, Clostridium perfringens, some strains of Staphylococcus aureus. 3) Gamma-hemolytic bacteria do not lyse red blood cells and they do not produce any halos around their colonies. They are non-pathogenic or opportunistic pathogens Examples: Enterococcus faecalis, Staphylococcus epidermidis, Streptococcus mutans

 Chapter 5 - Control of Microbial Growth

8.    [05-01] What are the differences between sterilization, disinfection, decontamination (sanitization)? – Lecture 2, Slide 19 –

Sterilization: Procedure that kills or removes all microbes, including endospores and viruses.
Disinfection:Kills/removes all pathogens. Non-pathogens and endospores may survive treatment. Achieved by two types of compounds disinfectants and antiseptics.

Pasteurization: Kills vegetative cells of pathogens and reduces the number of spoilage microbes. Does NOT affect endospores.

Decontamination (sanitization): Procedure that reduces overall number of microbes present. Some pathogens/opportunistic pathogens may survive treatment.

9.    [05-02] What is decimal reduction time and how does it affect the antimicrobial treatment? How does microbial number affect the antimicrobial treatment? – Lecture 2, Slide 20 –

D value (aka decimal reduction time)

- Time required to reduce population by 90%

- Depends on nature of microbe and antimicrobial procedure used

More resistant microbes present in sample = more longer/stringent antimicrobial treatment.

More microbes in sample = longer treatment

10. [05-03] Describe conditions and outcomes of boiling, pasteurization, and autoclaving. Which of these treatments can be considered as sterilization and why? – Lecture 2, Slide 22 –
Boiling: (100ºC for 5-10mins) Kills vegetative bacteria and viruses. However, most endospores survive. NOT considered sterilization.

Pasteurization: (Classical = 63ºC for 30mins; High Temp. Short Time HTST = 72ºC for 15 secs; Ultra high temperature = 140ºC for 2 secs)Kills vegetative bacterial cells. However, endospores and naked viruses survive → NOT sterilization.

Autoclaving: (121ºC at 15 psi for 20mins)Kills vegetative bacteria and endospores, viruses and all other microbes → Sterilization (BUT possible for prions to survive).

11. [05-04] What are conditions for hot air sterilization and autoclaving? Why does hot air sterilization take much longer time than autoclaving? – Lecture 2, Slides 22-23 –

Hot air sterilization: (170ºC for 2hrs or 200ºC for 1.5hrs) Material exposed to hot air for longer period of time b/c lack of pressure and moisture.
VS
Autoclaving: (121ºC at 15 psi for 20mins).

12. [05-05] Describe filtration and radiation as methods of antimicrobial treatment – Lecture 2, Slide 24 –

Filtration = Microbes mechanically removed from air/liquids if bigger than size of pores in filter. Not effective against viruses b/c they are so small.
Radiation = Various types ionizing (x-rays, gamma-rays), non-ionizing (UV light), microwaves. Carry large amount of energy that damages the proteins and nucleic acids through formation of highly reaction molecules and free radicals.

13. [05-06] Describe disk-diffusion method. Why should potential antimicrobial chemical be tested against various microbial groups? – Lecture 2, Slide 25 –

Disk-diffusion method: Used to test potential antimicrobials.

1) Paper disk is soaked in chemical being tested and overlaid over bacterial lawn (of chosen microbe).

2) Appearance of clear zone indicates the antimicrobial activity of tested compound. (Bigger clear zone = more effective compound is against microbe)

14. [05-07] Alcohols and ethylene oxide– their mechanism of antimicrobial action – Lecture 2, Slide 27 –

Alcohols (antiseptic) – kills bacteria and enveloped viruses, BUT not endospores/naked viruses. Mechanism: Dehydrates cells & dissolved lipids of plasma membrane and viral enveloped (has to be at least 60% alcohol, but less than 95%).

Ethylene oxide – Steriliant that kills all bacteria and other microbes including endospores, naked viruses. Mechanism: Very strong oxidant that damages proteins, nucleic acids and lipids.

15. [05-08] Halogens and phenolic compounds – their mechanism of antimicrobial action – Lecture 2, Slide 28 –

Halogens (Disinfectants/antiseptics) – Kills bacteria, but may/may not kill endospores. Mechanism: Strong oxidizers, denatures proteins and disturbs phospholipid layer.

Phenolic compounds (Disinfectants/antiseptics) –Kills mycobacteria, but may/may not kill endospores. Mechanism: Affects integrity of plasma membrane or viral envelopes and destroys proteins.