: Cultivation of Anaerobic Bacteria, Exercise# 11 & 12- MicroBio Lab

*Lab 27: Cultivation of Anaerobic Bacteria* Where does the Anaerobic bacteria grow in the Thioglycollate Broth Tube?

Only in the clear area below the red/pink area.

*Lab 27: Cultivation of Anaerobic Bacteria* What is the catalyst in the Brewer's Anaerobic Jar? What do they do?

Platidium Pellets They convert oxygen to water

The sodium thioglycolate in the broth creates a ________________________ in the tube.

Redox potential.

*Lab 27: Cultivation of Anaerobic Bacteria* What is the Indicator in the Thioglycollate Broth Tube? What colors will it turn and why?

Resazurin Aerobic = pink color when O2 is present Anaerobic = clear when O2 is not present

*Lab 27: Cultivation of Anaerobic Bacteria* What is this lab's purpose?

To determine the oxygen requirements of bacteria

Specialized methods are necessary to culture organisms _____________

anaerobically

*Lab 27: Cultivation of Anaerobic Bacteria* Facultative anaerobes do not show a ____________ for oxygen or an alternate form.

preference

What do the following classes of bacteria have in common? c) Facultative and Aerotolerant?

-Capable of living in the presence and absence of oxygen -Possess the enzymes necessary to neutralize toxin oxygen by-products

*Lab 27: Cultivation of Anaerobic Bacteria* Where does Aerobic bacteria grow in the Thioglycollate Broth Tube?

Above the red/pink area.

_______________ require atmospheric O2 (20%), and use O2 as the final electron acceptor in the electron transport system.

Aerobes

__________________ Grow equally well in the presence or absence of oxygen. They do possess enzymes necessary to neutralize toxic oxygen by-products, but the never use O2 as a final electron acceptor.

Aerotolerant Anaerobes

*Lab 27: Cultivation of Anaerobic Bacteria* What Jar creates an Anaerobic environment?

Brewer's Anaerobic Jar/ Gas Pak Jar

After examining the -Enterococcus faecalis - tube, was there a presence/absence of microbial growth and what was the location of growth?

Enterococcus faecalis is Aerotolerant anaerobe- grows throughout the entire tube

*Lab 27: Cultivation of Anaerobic Bacteria* _______________ systems allow facultative anaerobes to use free _____________ or some alternate oxygen source such as _________.

Enzyme oxygen nitrate

*Lab 27: Cultivation of Anaerobic Bacteria* The Candle Jar is used to reduce ______ concentration to between ______% and _______% by increasing ________.

O2 6% to 10 % CO2

_________________ cannot survive in the presence of any oxygen. They lack the enzymes necessary to break down the toxic by-products of oxygen. In these bacteria, the final electron acceptor in the electron transport system is a molecule other than O2.

Obligate anaerobes

What do the following classes of bacteria have in common? a) Obligate aerobes and Microaerophiles?

-Live in the presence of oxygen -Use oxygen as the final electron acceptor in the ETC

What cultures were used in this exercise (#11)?

-Staphylococcus xylosus -Mycobacterium smegmatis -Enterococcus faecalis -Micrococcus luteus -Saccharomyces cerevisiae -Clostridium sporogenes -Bacillus megaterium -Serratia marcescens

*Lab 27: Cultivation of Anaerobic Bacteria* Obligate Anaerobes require ______% O2.

0% / lack of O2

What are the 5 major bacterial oxygen classes?

1) Aerobes 2) Microaerophiles 3) Obligate anaerobes 4) Aerotolerant anaerobes 5) Facultative anaerobes

*Lab 27: Cultivation of Anaerobic Bacteria* What is the Thioglycollate Broth Tube made out of? (3)

1. Glucose 2. Cystine 3. Sodiuim Thioglycollate

*Lab 27: Cultivation of Anaerobic Bacteria* What media was used in this lab? (3)

1. Nutrient Agar Slants (4 per person) 2. Thioglycollate Broth Tubes (1 per person) 3. Nutrient Agar Plates (placed in Brewer's Anaerobic Jar)

*Lab 27: Cultivation of Anaerobic Bacteria* What microbes were used in this lab for all 3 media types? (4) What are their oxygen requirements?

1. Pseudomonas aeruginosa (Aerobe) 2. Escherichia coli (Facultative Anaerobe) 3. Enterobacter aerogenes (Facultative Anaerobe) 4. Clostridium sporogenes (Obligate Anaerobe)

*Lab 27: Cultivation of Anaerobic Bacteria* Results: Where did each microbe grow in the Thioglycollate Broth Tube?

1. Pseudomonas aeruginosa - grew at the top of the pink area (aerobe that requires oxygen) 2. Escherichia coli - all throughout the tube from red /pink area to clear area (facultative anaerobe, can grow w/ or without oxygen) 3. Enterobacter aerogenes - all throughout the tube from red/pink area to clear area (facultative anaerobe, can grow w/ or without oxygen) 4. Clostridium sporogenes - only below the red/pink area in the clear area because it is an obligate anaerobe

*Lab 27: Cultivation of Anaerobic Bacteria* Obligate Aerobes require ______% O2.

16%

*Lab 27: Cultivation of Anaerobic Bacteria* How does the reaction for breaking down toxic hydrogen peroxide look like?

2H + O2 -----> H2O , H2O2 (toxic) ---(catalase)---> H2O + O2

__________________ conditions are created by adding water to a gas generator envelope that is placed in the jar just before sealing.

Anaerobic

After examining the -Bacillus megaterium - tube, was there a presence/absence of microbial growth and what was the location of growth?

Bacillus megaterium is an Obligate Aerobe - grows at the top of the tube

*Lab 27: Cultivation of Anaerobic Bacteria* What kind of Jar creates a Microaerophilic environment?

Candle Jar

*Lab 27: Cultivation of Anaerobic Bacteria* What enzyme does aerobic bacteria have that anaerobic bacteria do not?

Catalase

After examining the -Clostridium sporogenes - tube, was there a presence/absence of microbial growth and what was the location of growth?

Clostridium sporogenes is an Obligate Anaerobe - grow at the bottom of the tube

________________________ are also able to live either in the presence or absence of oxygen, but they prefer oxygen so they can carry out aerobic respiration with O2 as final electron acceptor to maximize ATP yields. *These organisms can use other electron acceptors if O2 is not available, such as fumarate and nitrate. They can also utilize fermentative metabolism in the absence of oxygen.

Facultative Anaerobes

What do the following classes of bacteria have in common? b) Aerotolerant and Obligate anaerobes?

Final electron acceptor is a molecule other than oxygen -Capable of living in the absence of oxygen

*Lab 27: Cultivation of Anaerobic Bacteria* Where does the Facultative Anaerobic bacteria grow in the Thioglycollate Broth Tube?

Growth in the entire tube from red/pink area to the clear area.

*Lab 27: Cultivation of Anaerobic Bacteria* What is the indicator in the Brewer's Anaerobic Jar? What do its colors indicate?

Indicator Strip of Methylene Blue: Blue strip = aerobic conditions Clear strip = anaerobic conditions

*Lab 27: Cultivation of Anaerobic Bacteria* What does catalase do?

It breaks down the accumulation/buildup of toxic hydrogen peroxide during respiration

*Lab 27: Cultivation of Anaerobic Bacteria* After you add water (from the Platidium Pellets), what does the Gas Pak do?

It creates an anaerobic environment by releasing CO2 + H2 gases.

*Lab 27: Cultivation of Anaerobic Bacteria* What does the Sodium Thioglycollate component do in the Thioglycollate Broth Tube?

It lowers the oxidation / reduction potential (i.e. it makes the tube anaerobic which means that oxygen is removed)

*Lab 27: Cultivation of Anaerobic Bacteria* Where does the Microaerophilic bacteria grow in the Thioglycollate Broth Tube?

Just below the surface in the red/pink area.

______________ require O2 at below atmospheric concentrations, typically 2-10%. They have a limited ability to neutralize toxic oxygen, so excess O2 will kill the bacteria. However, they do use O2 as final electron acceptor in the electron transport system.

Microaerophiles

After examining the -Micrococcus luteus - tube, was there a presence/absence of microbial growth and what was the location of growth?

Micrococcus luteus is an Obligate aerobe - grows at the top of the tube

After examining the -Mycobacterium smegmatis - tube, was there a presence/absence of microbial growth and what was the location of growth?

Mycobacterium smegmatis is an Obligate Aerobe - so grows on the top

After examining the -Saccharomyces cerevisiae - tube, was there a presence/absence of microbial growth and what was the location of growth?

Saccharomyces cerevisiae is a microaerophile - grows towards the top (not exactly at the very top but close)

After examining the -Serratia marcescens - tube, was there a presence/absence of microbial growth and what was the location of growth?

Serratia marcescens is a facultative anaerobe - growth is spead throughout but is concentrated mostly at the top of tube

*Lab 27: Cultivation of Anaerobic Bacteria* Results: Which microbes showed growth in the Nutrient Agar Slants? Why?

Since the Nutrient Agar slants were an aerobic conditions... Growth: 1. Pseudomonas aeruginosa (blue/green) - grows in aerobic conditions 2. Escherichia coli (clear/white) - can grow in either aerobic or anaerobic conditions 3. Enterobacter aerogenes (clear/white) - can grow in either aerobic or anaerobic conditions No Growth: 4. Clostridium sporogenes - it is an obligate anaerobic which means it only grows strictly when there is no oxygen present

After examining the -Staphylococcus xylosus - tube, was there a presence/absence of microbial growth and what was the location of growth?

Staphylococcus xylosus is a Facultative Anaerobe- so it has some spread but the majority are toward the top of the tube.

*Lab 27: Cultivation of Anaerobic Bacteria* Results: Which microbes showed growth in the Nutrient Agar Plate? Why?

The Nutrient Agar Plates were placed in Brewer's Anaerobic Jar which creates an Anaerobic environment that lacks oxygen, therefore.... Growth: 1. Clostridium sporogenes - obligate anaerobe 2. Escherichia coli - facultative anaerobe (can grown w/ or without oxygen) 3. Enterobacter aerogenes - facultative anaerobe (can grow w/ or without oxygen) No Growth: 4. Pseudomonas aeruginosa - aerobe that only grows in the presence of oxygen

*Lab 27: Cultivation of Anaerobic Bacteria* What media is used in this lab?

Thioglycollate Broth Tube

Why is it important to avoid creating bubbles or aerating the liquefied BHI tubes when mixing the bacteria into the agar?

To avoid disturbing the oxygen gradient, if disturbed the results would not be accurate.

What is one way you can determine the oxygen requirements of a bacterium?

You can inoculate the bacterial culture into a melted agar deep tube of Brain Heart Infusion (BHI) media, mixing well to evenly distribute the bacteria throughout the liquified agar. Once the agar solidifies, an oxygen gradient is created -w/ atmospheric oxygen concentrations on the TOP and little to no oxygen present at the BOTTOM of the tube. The bacteria present will only be able to grow where their oxygen requirements are met and will localize to the area(s) of their oxygen requirements in the tube.

*Lab 27: Cultivation of Anaerobic Bacteria* Why does anaerobic bacteria get killed by the buildup of toxic hydrogen peroxide?

because it lacks the enzyme catalase that breaks it down

Bacteria have different oxygen requirements depending on the types of _________________ they possess.

enzymes

*Lab 27: Cultivation of Anaerobic Bacteria* Microaerophilic bacteria grow in the presence of ______ quantities of oxygen. Their O2 requirement is ______%.

minute 6% to 10%

*Lab 27: Cultivation of Anaerobic Bacteria* Most facultative anaerobes grow best in the presence of ___________.

oxygen

This reaction is catalyzed by the element _________________

palladium (#46 on element table)

What 2 chemical tablets are in the envelope?

sodium borohydride and sodium bicarbonate

*Lab 27: Cultivation of Anaerobic Bacteria* Facultative Anaerobes can grow _________ or ___________ O2 and require _______% O2.

with or without 0% to 21% O2