bahan untuk bakteri.docx

8/10/2019 bahan untuk bakteri.docx

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1. The image here shows three different genera of bacteria growing on EMB agar. They are

numbered 1 to 3. Which of the following conclusions is accurate?

a. Organism 1 is Gram-positive and

is unable to ferment glucose.

b. Organism 2 is Gram-negativeand can ferment sucrose.

c. Organism 3 is Gram-negative

and can ferment lactose*.

d. All three organisms are Gram-negative and can ferment

sucrose.

e. None of the above conclusions

are correct.

2. The product shown in the image here is used to identify

a. Gram-positive cocci.b. Gram-negative cocci.

c. Gram-positive bacilli .

d. Gram-negative bacilli. *

e. Gram-positive spirochetes.
http://www.mesacc.edu/~johnson/labtools/Dbiochem/ent5a.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/3emba.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/ent5a.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/3emba.jpg


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3. Staphylococcus aureus , a major pathogen, is usually

a. alpha hemolytic.b. beta hemolytic. *

c. gamma hemolytic.

d.

delta hemolytic.e. incapable of hemolysis.

4. These gelatin tubes have each been inoculated with a pure culture of different bacteria.

The tubes were incubated for 48 hours and have been refrigerated for 30 minutes. The

arrow in this image indicates an organism that is

a. capable of degrading gelatin. *

b. incapable of degrading gelatin.

c. capable of clotting plasma.d. incapable of clotting plasma .

e. capable of degrading hydrogen

peroxide.

5. These tryptone broths were each inoculated with a different bacteria. After incubation

Kovac's reagent was added to both tubes. Bacteria that produce the result shown at the

arrow in this image are
http://www.mesacc.edu/~johnson/labtools/Dbiochem/gela.jpg


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a. Voges-Proskauer positive.b. Voges-Proskauer negative.

c. indole positive. *

d. indole negative.e. methyl red positive.

6. The bacteria that produced the result shown at the arrow in this image

a.

produce very acid products fromglucose.b. produce somewhat alkaline

products from glucose.

c. produce indole when it

degrading trptophan.d. do not produce indole when

degrading trptophan.

e. are able to utilize citrate as theirsole carbon and energy source .*

7. The media shown in this image is mannitol salt agar. The organism that the arrow is

pointing to is
http://www.mesacc.edu/~johnson/labtools/Dbiochem/indolea.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/cita.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/indolea.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/cita.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/indolea.jpg


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a. able to ferment mannitol and

probably is Staphylococcus

epidermidis.

b. able to ferment mannitol and


aureus.c. unable to ferment mannitol and


epidermidis .*d. unable to ferment mannitol and


aureus.

e. able to ferment glucose andprobably is Staphylococcus

aureus.

8. This image shows the results of bacteria inoculated into motility media. The organism in

tube 2

a. is motile and possesses flagella.

*

b. is nonmotile and lacks flagella.c. is motile and lacks flagella.

d. is nonmotile and possesses

flagella.

9. Methyl red test results for different organisms are shown in this image. Which

conclusion is correct?
http://www.mesacc.edu/~johnson/labtools/Dbiochem/msaa.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/mota.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/msaa.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/mota.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/msaa.jpg


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a. Tube 1 indicates an acidic pH as

a result of fermentation of

lactose.

b.

Tube 2 indicates an acidic pH asa result of fermentation of

lactose.

c. Tube 1 indicates an acidic pH asa result of fermentation of

glucose.

d. Tube 2 indicates an acidic pH as

a result of fermentation ofglucose. *

e. None of the above are correct

conclusions for these results.

10. Pathogens that are usually beta-hemolytic include

a. Staphylococcus aureus.b. Streptococcus pyogenes(Group A Strep).

c. Streptococcus pneumoniae.

d. a and b only *e. a, b and c
http://www.mesacc.edu/~johnson/labtools/Dbiochem/mra.jpghttp://www.mesacc.edu/~johnson/labtools/Dbiochem/mra.jpg


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Catalase test

Posted by:famsbcon:July 30, 2009

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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 like catalase and superoxidase dismutasemust

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

requires it to be removed instantly??
http://famsbc.wordpress.com/2009/07/30/catalase-test/aerobes-anaerobes-2/


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answer: H2O2.

Functions of catalase

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 H2O2down into water and O2, the presence of oxygen can be characterizedby 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 toprevent oxygen accumulation. Why?

Because since oxygen is totally not used for survivalof these organisms, they do not have theabilityto produce catalase.

e.g. Clostridium, Lactobacillus, Streptococcus

Carbohydrate Oxidation-Fermentation

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Fermentation can occur in the presenceof oxygen orabsence of oxygen.

If bacteria utilises carbohydrates for nutrients, there may be2 end products, a gas and acid. Thesubstrate formed from the metabolism of carbohydrate is either glucose or lactose.

Even if bacteria releases enzymes that enable to use carbohydrates through fermentation andoxidation, gas may or may not be produced.

FERMENTATION is noted by acid production which can be observed by a colour changein

Durham tubesaka carbohydrate fermentation tube.

Phenol Redindicator is red in neutral or alkaline solution

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

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

inverted tube inside the bigger tube. Therefore,gas is producedfrom the breakdown ofcarbohydrates.
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Alfred.E.Brown. (2007). Bensonss microbiological applications: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Johnson, T.R., & Case, C.L. (2007). Laboratory experiments in mi crobiology. (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 hasoccured?

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 reddue to NH3 production.
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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.

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

carbohydrate into glucose & lactose.

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

large amounts of acidand organisms that only produce neutral content(acetoin)

M-Methyl Red. VP- Voges -Proskauer. Methyl Red is different from Phenol Red that wemention 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 ofMethyl Red. REDremains.

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

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

http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/methyl-red-te3st-2/http://famsbc.files.wordpress.com/2009/07/table-of-durham-fermentation.png?w=300http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation/methyl-red-te3st-2/http://famsbc.files.wordpress.com/2009/07/table-of-durham-fermentation.png?w=300


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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 turnslight brown, it is a (-) result.

Please note that production of acetoin is also affected by the duration of incubation, hence, falsenegative results may be observed.

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

citrate. When citric acid or sodium citrate is in solution, it loses a protonor sodium ion to forma 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 toblue when medium turnsalkaline, whichmeans (+) result.

Why alkaline means citrate is utilised by the bacteria?

When bacteria uses citrate(carbon) and ammonium(nitrogen), medium turns alkaline as ammoniais produced from ammonium.
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Johnson, T.R., & Case, C.L. (2007).Laboratory experiments in mi crobiology. (8th ed.). San Francisco: Pearson Education.

Citrobacter, Enterobacterwill display (+) result while E. coli& Klebsiellawill display (-) result.

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

formed during metabolism. For example, Hydrogen Sulphide.

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


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Oxidative-Fermentative Metabolism


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

we will test with an glucose OF oxidative- fermentative agar. If agar turns yellow, acidisproduced. If agar turns green, no acidis 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.

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

conditions.

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Nitrogen metabolismurea Hydrolysis


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UREASE breaks down urea into ammonia

(NH3) & carbon dioxide (CO2).

Alfred.E.Brown. (2007). Bensonssmicrobiological appli cations: laboratory manual in general microbiology. (10th ed.). New York: Mc Graw Hill.

Properties of urea medium

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

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

Proteus vulagrisis one bacteria that produces urease to break down urea.

Tryptophan Degradation


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Tryptophan is degraded bytryptophanaseinto indole, ammonia, pyruvic acid. Pyruvic acid 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 ringat the top of the agar/ broth is formed whenKovacs reagent reacts with indole. This is the (+) result.

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

produced.


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)
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17/22

Lipolysis


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Lipolysis is carried out by LIPASES.

Triglycerides(big molecules derived from lipids) are broken down into fatty acids & glycerol

If 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) OR2. oil droplets (when tributyrin is not completely broken down into fatty acids)

Organism responsible for exhibiting (+) result is Staphyloccocus aureus.

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

Properties of Spirit Blue Agar

Contents- tributyrin (simple animal triglyceride)
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Tributyrin acts as a substrate for exoenzyme lipase.

Protein Catabolism (Proteolysis)


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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- ureahydrolysis)

-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 ofagar indicates a (+) result.
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Organism that gives (+) result isBacillus subtilis, Clostridiumspieces. (-) 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 oflactobacilli.

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 caseinin 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 gelatinaseinto smaller polypeptides, peptones and amino acids that cancross 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.
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Johnson, T.R., & Case, C.L. (2007).Laboratory experiments in mi crobiology. (8th ed.). San Francisco: Pearson Education.

Hence, a (+) result is indicated by the liquid stateof gelatin.Bacillus subtilisis able to produceproteolytic 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 whenHydrogen Sulphide is release.

Why? Some bacteria have the ability to give off H2S from sulphur containing amino acidsafter 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.
http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/gelatin-hydrolysis/


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

that produces (+) are Citrobacter, Salmonella.
http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/kligers-fe-agar-w-fermentation-h2s-productn-gas-acid/http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/triple-sugar-iron-agar/http://famsbc.wordpress.com/2009/07/25/protein-hydrolysis-proteolysis/kligers-fe-agar-w-fermentation-h2s-productn-gas-acid/


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Title: Motility test

Description(from left to right)

Uninoculated control

Pseudomonas fluorescens(motile; obligate aerobe)

Staphylococcus epidermidis(nonmotile; facultative anaerobe)

Escherichia coli(motile; facultative anaerobe)

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