oxygen is sometimes toxic

8/3/2019 Oxygen is Sometimes Toxic

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Oxygen is sometimes toxic.

Small amounts of superoxide free radicals are formed during the normal respiration of

organisms that use oxygen as the final electron acceptor.

Obligate anaerobes from some oxygen free radicals that are toxic to the cell. Hence, if bacteria

wants to grow in oxygen environment, enzymes likecatalase and superoxidase

dismutase must be present for neutralization of the toxic form of oxygen(oxygen

radical)


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During normal aerobic respiration, hydrogen ions are produced and have to be removed by

bacterial cell. The electron transport system (ETS) in cellular respiration (a part of glycolysis)

involves these H+ ions and combines them with oxygen to form water. Water is harmless.

Energy is given off and stored in the form of Adenosine Triphosphate.

What is toxic is Hydrogen Peroxide that is formed by the cytochromes in ETS. Water being

harmless is not required to be removed by the bacteria. So, what is harmful to bacteria cell

that requires it to be removed instantly??

answer: H2O2.

Functions of catalase
http://famsbc.wordpress.com/2009/07/30/catalase-test/aerobes-anaerobes-2/


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Protects bacteria from toxic hydrogen peroxide (H2O2) accumulation, which can

occur during aerobic metabolism. If hydrogen peroxide accumulates, it becomes toxic to

the organism.

Since Catalase breaks H2O2 down into water and O2, the presence of oxygen can be

characterized by bubbles which indicates a (+) result.

What bacteria could mostly likely be detected?

most aerobic organism make catalase.http://student.ccbcmd.edu/courses/bio141/labmanua/lab8/catstaph.html

Most aerobic organsims will display (+) results.

e.g. Staphyloccocus aureus.

Some anaerobic organisms will display (-) results, indicating that they do not produce catalase

to prevent oxygen accumulation. Why?

Because since oxygen is totally not used for survival of these organisms, they do not have

the ability to produce catalase.e.g. Clostridium, Lactobacillus, Streptococcus

Carbohydrate Oxidation-FermentationPosted by: famsbc on: July 28, 2009

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Fermentation can occur in the presence of oxygen or absence of oxygen.

If bacteria utilises carbohydrates for nutrients, there may be 2 end products, a gas and acid.

The substrate formed from the metabolism of carbohydrate is either glucose or lactose.

Even if bacteria releases enzymes that enable to use carbohydrates through fermentation and

oxidation, gas may or may not be produced.

FERMENTATION is noted by acid production which can be observed by acolour

change in Durham tubes aka carbohydrate fermentation tube.

Phenol Red indicator is red in neutral or alkaline solution

Ifacid is present(+), phenol red changes from red > yellow

If there is a small space at the top of the small tube, it means that gas is trapped in the

small inverted tube inside the bigger tube. Therefore, gas is produced from the

breakdown of carbohydrates.

Alfred.E.Brown. (2007). Bensonss microbiological applications: laboratory manual in generalmicrobiology. (10th ed.). New York: Mc Graw Hill.
http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/durhamn-fer-tub-w-gas-present/http://famsbc.files.wordpress.com/2009/07/glycolysis1.png?w=300


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Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in microbiology. (8th ed.). San Francisco:Pearson Education.

Look at the 1st picture above, why is it red if gas is produced and it just means fermentation

has occured?

Due to prolonged incubation periods(more than 24h), bacteria will begin to grow

oxidatively(oxygen dependent) on the peptone contents of the fermentation medium after

using up the carbohydrate contents, causing the neutralization of phenol red indicator and

turing it red due to NH3 production.

The only organism that has the ability to break down carbohydrate into glucose and lactose

is E.coliand it also has the ability to produce gas during fermentation.

For P. aerguinosa, it displayed negative results for all, which means it cant break down

carbohydrate into glucose & lactose.

Another test which is the MRVP test is able to differentiate between organisms that produce

large amounts ofacid and organisms that only produce neutral content(acetoin)

M-Methyl Red. VP- Voges -Proskauer. Methyl Red is different from Phenol Red that we mention

earlier. It has a pH of 4.4-6. Hence, it changes colour at this range.For MR test, if organic acid is produced here, the (+) result is just a no change in

colour of Methyl Red. RED remains.
http://famsbc.files.wordpress.com/2009/07/table-of-durham-fermentation.png?w=300http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/carbofermentation/


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if neutral acetoin is produced here, the (-) result is a change in colour

fromRED to YELLOW because at pH 7, the indicator will change its colour.


To test for the presence of acetoin, we use VP test where potassium hydroxide and naphthol

are added.

The upper of the medium will turn red. (+) If the medium turns light brown, it is a (-) result.

Please note that production of acetoin is also affected by the duration of incubation, hence,

false negative results may be observed.

We can use Kosers citrate or Simmons citrate to test for the ability of bacteria

to ferment citrate. When citric acid or sodium citrate is in solution, it loses a proton or

sodium ion to form a citrate ion. Bacteria with citrate lyase can break down citrate to form

pyruvate. Pyruvate can be further reduced in fermentation.

Purpose of performing the citrate test

Tests for the ability of bacteria to convert citrate (an intermediate of the Krebs cycle)into oxaloacetate (another intermediate of the Krebs cycle)

Contents of Simmon citrate include

sodium citrate as the carbon source

monoammonium phosphate as the nitrogen source

and bromthymol blue indicator that changes to blue when medium turnsalkaline,

which means (+) result.

Why alkaline means citrate is utilised by the bacteria?

When bacteria uses citrate(carbon) and ammonium(nitrogen), medium turns alkaline as

ammonia is produced from ammonium.
http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/methyl-red-te3st-2/


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Citrobacter, Enterobacterwill display (+) result while E. coli& Klebsiellawill display (-) result.
http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/citrate-test/


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http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/enterbacteriaceae-family/


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After knowing that some bacteria utilises citrate, we can also find out if other products are

formed during metabolism. For example, Hydrogen Sulphide.


Oxidative-Fermentative MetabolismPosted by: famsbc on: July 27, 2009

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How do we determine if type of metabolism is fermentative or oxidative?

we will test with anglucose OF oxidative- fermentative agar. If agar turns yellow, acidis

produced. If agar turns green, no acid is produced. Motility of the bacteria can also be

determined by the presence of turbidity (cloudiness)

OXIDATIVE metabolism may or may not cause an acid to be produced for aerobic conditions.

No acid will be produced for anaerobic conditions.
http://famsbc.wordpress.com/2009/07/27/oxidative-fermentative-metabolism/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/27/oxidative-fermentative-metabolism/#respondhttp://famsbc.wordpress.com/2009/07/27/oxidative-fermentative-metabolism/ecoli-eaerogenes-controls-included-of-test/http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/hydrogen-sulphide-prod/http://famsbc.wordpress.com/2009/07/27/oxidative-fermentative-metabolism/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/27/oxidative-fermentative-metabolism/#respond


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FERMENTATIVE metabolism will cause an acid to be produced for aerboic and anaerobic

conditions.

Alfred.E.Brown. (2007). Bensonss microbiological applications: laboratory manual in general

microbiology. (10th ed.). New York: Mc Graw Hill.

Nitrogen metabolism urea HydrolysisPosted by: famsbc on: July 27, 2009

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UREASE breaks down urea intoammonia (NH3) & carbon dioxide(CO2).
http://famsbc.wordpress.com/2009/07/27/urea-hydrolysis/http://famsbc.wordpress.com/2009/07/27/urea-hydrolysis/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/27/urea-hydrolysis/#respondhttp://famsbc.wordpress.com/2009/07/27/oxidative-fermentative-metabolism/table-of-oxidation-and-fermentation/http://famsbc.wordpress.com/2009/07/27/oxidative-fermentative-metabolism/of-test/http://famsbc.wordpress.com/2009/07/27/urea-hydrolysis/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/27/urea-hydrolysis/#respond


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Properties of urea medium

Since theres phenol red pH indicator, pH indicator changes from yellow to bright pink if NH3 is

produced.

At more than pH 6.8, the colour change represents a (+) result

Proteus vulagris is one bacteria that produces urease to break down urea.

Tryptophan DegradationPosted by: famsbc on: July 25, 2009

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Tryptophan is degraded by tryptophanase into indole, ammonia, pyruvic acid. Pyruvicacid is then involved in metabolic pathway so that ATP energy for bacterial cell is generated.

While other products like NH3 and Pyruvate is metabolised, indole is not. Hence, it stays in the

medium.

Upon addition of Kovacs reagent, deep red ring at the top of the agar/ broth is formed

when Kovacs reagent reacts with indole. This is the (+) result.

E.coliis one of the organism that can display (+) result since exoenzyme tryptophanase is

produced.
http://famsbc.wordpress.com/2009/07/25/tryptophan-degradation/http://famsbc.wordpress.com/2009/07/25/tryptophan-degradation/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/tryptophan-degradation/#respondhttp://famsbc.wordpress.com/2009/07/27/urea-hydrolysis/urease-test/http://famsbc.wordpress.com/2009/07/25/tryptophan-degradation/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/tryptophan-degradation/#respond


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Properties of Tryptic Soy Broth/Agar

-tryptone is one of the contents of TSB/ TSA and is derived from casein.(recall protein from

protein hydrolysis)

LipolysisPosted by: famsbc on: July 25, 2009

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Lipolysis is carried out by LIPASES.Triglycerides(big molecules derived from lipids) are broken down into fatty acids & glycerolIf pH of medium, used for testing whether lipase is present to break down lipids, is lowered.

It indicates acidic product (fatty acid) is formed.

Trigylceride used here is tributyrin that can be found in medium Spirit Blue Agar.

There are 2 indicators for (+) result.

1. dark blue precipitate(tributyrin is completely broken down into fatty acids) OR

2. oil droplets (when tributyrin is not completely broken down into fatty acids)

Organism responsible for exhibiting (+) result is Staphyloccocus aureus.
http://famsbc.wordpress.com/2009/07/25/lipolysis/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/lipolysis/#respondhttp://famsbc.wordpress.com/2009/07/25/tryptophan-degradation/tryptophan-hydrolysis/http://famsbc.wordpress.com/2009/07/25/lipolysis/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/lipolysis/#respond


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Alfred.E.Brown. (2007).Bensonss microbiological applications: laboratory manual in generalmicrobiology. (10th ed.). New York: Mc Graw Hill.

Properties of Spirit Blue Agar

Contents- tributyrin (simple animal triglyceride)

Tributyrin acts as a substrate for exoenzyme lipase.

Protein Catabolism (Proteolysis)Posted by: famsbc on: July 25, 2009

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Proteolysis is carried out by

PROTEASE.In the following posts we will mention

- bacterias ability to hydrolyse casein, gelatin

- urea hydrolysis by detecting presence of urease(refer to post on nitrogen metabolism- urea

hydrolysis)

-hydrogen sulphide production

Casein(a protein) is broken down by protease into peptones and amino acids.

During the degradation process, polypeptide bonds are broken.

Once the bonds are broken, amino acids are produced. A clear zonesurrounding streak line

of agar indicates a (+) result.
http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/#respondhttp://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/skim-milk-11/http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/skim-milk-10/http://famsbc.wordpress.com/2009/07/25/lipolysis/fat-hydrolysis/http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/#respond


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Organism that gives (+) result is Bacillus subtilis, Clostridium spieces. (-) results are indicative

that organismdoes not cause a clear zone. an example of organism displaying (-) result

is Escherichia coli.Alfred.E.Brown. (2007). Bensonss microbiological applications: laboratory manual in general

microbiology. (10th ed.). New York: Mc Graw Hill.

Properties of skim milk agar

Skim milk agar contents- casein, lactose and other nutrients, which support growth of

lactobacilli.

Gives the white colour to milk.

As seen from the above, we can infer if bacteria present in milk is able to break down the

casein in milk, milk may have abnormal particles that are not white in colour.

Application of this biochemical this is in food testing.

Another protein commonly found in food products is gelatin.

It is broken down by gelatinase into smaller polypeptides, peptones and amino acids that can

cross the cell membrane and be utilised by the organism.

Property of Gelatin agar

INTERESTING to note: when gelatin is broken down via hydrolysis, it cannotsolidify anymore,

the areas of solid gelatin media where the organsim grows, will turn into liquid. Even if you

refrigerate this medium, the media will remains liquid.
http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/gelatin-hydrolysis/http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/casein-hydrolysis/


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Hence, a (+) result is indicated by the liquid state of gelatin. Bacillus subtilisis able to

produce proteolytic exoenzyme gelatinase to give the (+) result.

Why is gelatin not used widely as a selective media for isolating bacteria? (hint: what is

gelatin agar unique property?)

Most bacteria do not contain enzymes that liquefy gelatin. Hence, it is not uselfy for isolatingmicrobes for bacterial identification.

Decomposition of amino acid cysteine is detected by the formation of ferrous

sulphide when Hydrogen Sulphide is release.

Why? Some bacteria have the ability to give off H2S from sulphur containing amino

acids after proteins are broken down into amino acids by enzymes.

When H2S is produced, sulfide ion reacts with the metal salt to product a black

precipitate(+) result.


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http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/triple-sugar-iron-agar/


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LOOK AT TUBE 3. the black ppt indicates that Hydrogen Sulphide was produced. (+). Bacteria

that produces (+) are Citrobacter, Salmonella.

Starch HydrolysisPosted by: famsbc on: July 25, 2009

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Reaction carried out by AMYLASE isstarch hydrolysis

Reagent required to be added

Iodine (pH indicator)

Starch + iodine > starch- iodine complex

Undegraded starch reacts with Iodine to form a dark blue starch- iodine complex that covers

the entire agar. (-)

Escherichia coli

Ifstarch(polysaccharide) is broken down into glucose(or any other monosaccharides/ di

saccharides), glucose will then react with iodine, forming a clear zone surrounding streak

line. (+)

Organism that gives (+) results

Bacillus substilis

http://famsbc.wordpress.com/2009/07/25/starch-protein-lipid-hydrolysis-and-urea-tryptophan-degradation/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/starch-protein-lipid-hydrolysis-and-urea-tryptophan-degradation/#respondhttp://famsbc.wordpress.com/2009/07/25/starch-protein-lipid-hydrolysis-and-urea-tryptophan-degradation/starch-hydrolysis-2/http://famsbc.wordpress.com/2009/07/25/starch-protein-lipid-hydrolysis-and-urea-tryptophan-degradation/http://famsbc.wordpress.com/category/uncategorized/http://famsbc.wordpress.com/2009/07/25/starch-protein-lipid-hydrolysis-and-urea-tryptophan-degradation/#respond


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Here are other photos of starch hydrolysis test where drops of bacteria culture were placed

onto the agar medium

http://www.microbiologyatlas.kvl.dk/biologi/english/showbio.asp?articleid=plade2

Properties of starch agar are related to its contents like beef extract, soluble starch, and agar

Beef extract provides the nitrogen, vitamins, carbon and amino acids, while agar is the

solidifying agent.

It is a differential medium used to determine whether a bacteria is able touse starch as a

carbon source and an energy source.

Introduction to biochemical testsPosted by: famsbc on: July 25, 2009

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Purpose of Biochemical Tests
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Makes use ofenzymaticactivities todifferentiateamong bacteria.

It does not mean if 1 bacteria in the same bacterial group can ferment carbohydrate, all

others will.

Examples of biochemical reactions are oxidation, fermentation, hydrolysisand degradation.

*in later parts of the posts, u will come across terms like proteolysis, lipolysis. So what dothey mean? lysis= breakdown.

Proteo = protein. Lipo = Lipid

Products of biochemical reactions cause changes to the medium that you have inoculated the

organism with.

E.g. an acidic productpH of a medium.

Bacteria have a rigid cell wall. This cell wall causes the bacteria to be unable to surround and

eat their food through phagocytosis. Bacteria uses metabolism to gain nutrients.

pH indicator in the medium will exhibit a color change indicating that an exoenzyme isreleased by a bacteria that cause the product to be formed.

How do bacteria obtain nutrients then? excretion of exoenzymes

exo= outside. exoenzymes- enzymes outside cell that break down large molecules

macromolecules are broken down into smaller units

smaller units can enter bacterial cell for metabolism

Properties of enzymes

Larry McKane., & Judy Kandel. (1996). Microbiology: essentials and applications. (2nd ed.). NewYork: McGraw Hill.


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Simple enzymes are composed entirely of proteins.

Larry McKane., & Judy Kandel. (1996). Microbiology: essentials and applications. (2nd ed.). New

York: McGraw Hill.

Complex enzymes contain additional non protein molecules tightly associated with

the protein.

Tightly associated accessory molecules are referred to as prosthetic groups.

Enzymes are highly specific in their catalytic action.

In general, an enzyme works like a jigsaw. Itrecognizes only 1 set of

substrate. Substrateis the material on which enzyme acts. After enzyme acts, substrate is

converted to one particular set of products.

Enzyme has an area known as the active sitethat reacts with the substrate. The substratemust fit nicely into the active site.

If shape does not fit into the jigsaw(enzyme), nothing happens.

Some enzymes require small molecules to help carry out their catalytic

role. Without these small helper molecules, such enzymes are inactive. There are

other helper molecules that are temporary parts of enzyme. These temporary

parts assist in enzyme- mediated reaction. They can be organic

molecules(coenzymes) or metals that function as cofactors.

Coenzymes can be electron carriers NAD, NADP, FAD.

DO you know how to identify enzyme?

Enzyme usually ends with a ase

Oxidase

Urease Gelatinase

Catalase

Lipase

Dehydrogenase

Protease

Functions of enzymes

Marcromolecules are degraded.

Does it mean that small molecules will not be degraded?
http://famsbc.wordpress.com/2009/07/25/purpose-of-biochemical-tests/jigsaw/


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NO! small molecules like tryptophane are degraded so that they can acquire carbon

compounds to prepare for metabolism.
http://famsbc.wordpress.com/2009/07/25/purpose-of-biochemical-tests/table-of-micromolecules-2/http://famsbc.wordpress.com/2009/07/25/purpose-of-biochemical-tests/table-of-macromolecules-2/

oxygen is sometimes toxic

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