Identification of unknown Bacteria

Have you ever thought about how to find an unknown bacteria among thousands of a microbiom? Or what's the general methodology to reach the right result in Bacterial diagnostics?

Or have you ever been confused when test all acknowledged protocols as a routine in laboratory but you still not sure to tell the right choices? (when there is no PCR or Antibodies to easily detect your targets before coffee time! )

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Published by McGraw-Hill 2015, ISBN 978-0-07-340241-3

Here is the briefing by mindjet-maps, although it's not perfect but definitely is a good start point to get more focus on logical diagnostics (instead of trying all catalog numbers of product lists which sometimes looks like blind culturing to find answers besides contamination high-risk made by several handling without preserving stock sample, logically!!!

Here is MindMaping pictures that you can find high quality as the link below (my Google Drive)... hope it be useful for students as it was needed for me along bachelor's degree before...

https://drive.google.com/open?id=1nlVos-9RnstIswxdc08-XM-tkOccIHQU

1, Identification of Unknown Bacteria 

 1.1, What we have to look for 

   1.1.1, organisms’ morphological characteristics 

   1.1.2, cultural characteristic s 

   1.1.3, physiological (biochemical) characteristics

 1.2, organisms’ morphological characteristics 

   1.2.1, Make the unknown broth culture 

     1.2.1.1, two nutrient agar slants from deep of broth culture

       1.2.1.1.1, 20°c for 24h

         1.2.1.1.1.1, Select the best growth tube as the reserve stock 

       1.2.1.1.2, 37°c for 24h

         1.2.1.1.2.1, Use other tubes as the working stock 

           1.2.1.1.2.1.1, Nutrient broth

             1.2.1.1.2.1.1.1, Incubate at the optimum temperature for 24 hours

               1.2.1.1.2.1.1.1.1, Do gram staining + measure the size

               1.2.1.1.2.1.1.1.2, perform the proper motility tests: Semisolid medium (for pathogens)

                 1.2.1.1.2.1.1.1.2.1, For pathogens, SIM medium can be used to ascertain motility

                   1.2.1.1.2.1.1.1.2.1.1, In general, cocci are nonmotile, whereas rods can be either motile or nonmotile

               1.2.1.1.2.1.1.1.3, Wet mount slide

Gram stain: Make a Gram-stained slide from the slant and compare it with slide made from nutrient broth.

If organism is a nonpathogen, make a wet mount or hanging drop slide

                 1.2.1.1.2.1.1.1.3.1, For nonpathogens, the wet mount or hanging drop prepared from a broth culture is the preferred way to determine motility

                   1.2.1.1.2.1.1.1.3.1.1, In general, cocci are nonmotile, whereas rods can be either motile or nonmotile; write all information in descriptive chart.

           1.2.1.1.2.1.2, Nutrient agar slant

             1.2.1.1.2.1.2.1, Incubate at the optimum temperature for 24 hours (use it for especial staining and tests)

               1.2.1.1.2.1.2.1.1, Gram stain: Make a Gram-stained slide from the slant and compare it with slide made from nutrient broth

                 1.2.1.1.2.1.2.1.1.1, Note: The results from the Gram stain can be verified by performing the following test: Place one drop of 3% KOH on a microscope slide and transfer a loopful of your unknown cells to the KOH solution. While observing the slide edge-on at eye level, mix the cells and KOH solution and slowly raise the loop from the cells. Gram-negative cells will lyse in the KOH solution, releasing their DNA and causing the liquid to become very viscous

                 1.2.1.1.2.1.2.1.1.2, Keep in mind that short rods with round ends (coccobacilli) look like cocci. If you have what seems to be a coccobacillus, examine many cells before you make a final decision.

                 1.2.1.1.2.1.2.1.1.3, Also, keep in mind that while rod-shaped organisms frequently appear as cocci under certain growth conditions, cocci rarely appear as rods. (Streptococcus mutans is unique in forming rods under certain conditions.)

                 1.2.1.1.2.1.2.1.1.4, It is generally safe to assume that if you have a slide on which you see both coccus-like cells and short rods, the organism is probably rod-shaped. This assumption is valid, however, only if you are not working with a contaminated culture!

               1.2.1.1.2.1.2.1.2, Simple stain: Use Loeffler's methylene blue if metachromatic granules are suspected

                 1.2.1.1.2.1.2.1.2.1, VERY IMPORTANT TIP:

?IF LOEFFLER'S METHYLEN BLUE UPKEEPS BY LIVING BACTERIA TO STAIN METACHROMATIC GRANULES, SO WE CAN USE THIS SOLUTION TO DIFFERENTIATE LIVE AND DEATH CELLS AND SO THEN HAVING GOOD COUNT WHEN ENUMERATION TEST NEEDED!!! 

               1.2.1.1.2.1.2.1.3, Spore stain: If the organism is a grampositive rod, do a spore stain

                 1.2.1.1.2.1.2.1.3.1, If your unknown is a gram-positive rod, it may be an endospore-former. Endospores, however, do not usually occur in cocci or in gram-negative rods. Examination of your Gram-stained slide made from the agar slant should provide a clue since endospores show up as transparent oval structures in Gram-stained preparations. Endospores can also be seen on unstained organisms if studied with phase-contrast optics

                 1.2.1.1.2.1.2.1.3.2, Since some spore-formers require at least a week’s time of incubation before forming spores, it is prudent to double-check for spores in older cultures

               1.2.1.1.2.1.2.1.4, Acid-fast stain: If the organism is a gram-positive rod, make an acid-fast stained slide

                 1.2.1.1.2.1.2.1.4.1, The mycobacteria and some species of Nocardia are acid-fast. For these bacteria, the presence of acidfastness can interfere with the Gram stain, causing these bacteria to stain gram-positive. Performing the acid-fast stain will sort out part of this problem

                 1.2.1.1.2.1.2.1.4.2, Do not depend solely on the Gram stain as the results can be misleading, especially for the acid-fast bacteria

                 1.2.1.1.2.1.2.1.4.3, If your unknown is a gram-positive, non–sporeforming rod, it could be an acid-fast bacterium. Acidfastness can vary with culture age, but most cultures display this property after 2 days of incubation. For best results, do not do this stain on old cultures

               1.2.1.1.2.1.2.1.5, Blood agar culturing 

                 1.2.1.1.2.1.2.1.5.1, 37°c for 24h

                   1.2.1.1.2.1.2.1.5.1.1, Although a capsule stain, may be performed at "Nutrient agar slant" colonies, it might be better to wait until a later date when you have the organism growing on blood agar. Capsules usually are more apparent when the organisms are grown on Blood agar medium

   1.2.2, Report sheet (brief of flowcharts)

     1.2.2.1, Sample report sheet

       1.2.2.1.1, MORPHOLOGICAL CHARACTERISTICS

         1.2.2.1.1.1, Cell shape

         1.2.2.1.1.2, Arrangement

         1.2.2.1.1.3, Size

         1.2.2.1.1.4, Spores

         1.2.2.1.1.5, Gram’s Stain

         1.2.2.1.1.6, Motility

         1.2.2.1.1.7, Capsules

         1.2.2.1.1.8, Special Stains

       1.2.2.1.2, CULTURAL CHARACTERISTICS

         1.2.2.1.2.1, Colonies

           1.2.2.1.2.1.1, Nutrient Agar

           1.2.2.1.2.1.2, Blood Agar

         1.2.2.1.2.2, Agar Slant

         1.2.2.1.2.3, Nutrient Broth

         1.2.2.1.2.4, Gelatin Stab

         1.2.2.1.2.5, Oxygen Requirements

         1.2.2.1.2.6, Optimum Temp.

       1.2.2.1.3, PHYSIOLOGICAL CHARACTERISTICS

         1.2.2.1.3.1, Fermentation

           1.2.2.1.3.1.1, Glucose

           1.2.2.1.3.1.2, Lactose

           1.2.2.1.3.1.3, Sucrose

           1.2.2.1.3.1.4, Mannitol

         1.2.2.1.3.2, Hydrolysis

           1.2.2.1.3.2.1, Gelatin Liquefaction

           1.2.2.1.3.2.2, Starch

           1.2.2.1.3.2.3, Casein

           1.2.2.1.3.2.4, Fat

         1.2.2.1.3.3, IMViC

           1.2.2.1.3.3.1, Indole

           1.2.2.1.3.3.2, Methyl Red

           1.2.2.1.3.3.3, Voges-Proskauer (acetylmethylcarbinol)

           1.2.2.1.3.3.4, Citrate Utilization

           1.2.2.1.3.3.5, Nitrate Reduction

           1.2.2.1.3.3.6, H2S Production

           1.2.2.1.3.3.7, Urease

           1.2.2.1.3.3.8, Catalase

           1.2.2.1.3.3.9, Oxidase

           1.2.2.1.3.3.10, DNase

           1.2.2.1.3.3.11, Phenylalanase

       1.2.2.1.4, Reactions 

         1.2.2.1.4.1, Litmus Milk

           1.2.2.1.4.1.1, Acid

           1.2.2.1.4.1.2, Alkaline

           1.2.2.1.4.1.3, Coagulation

           1.2.2.1.4.1.4, Reduction

           1.2.2.1.4.1.5, Peptonization

           1.2.2.1.4.1.6, No Change

 1.3, Cultural characteristics (It means, macroscopic appearance on different kinds of media.)

   1.3.1, First period (Inoculations) ((by using of original broth culture of your unknown))

     1.3.1.1, one nutrient agar plate

       1.3.1.1.1, Pour a petri plate of nutrient agar for each unknown and streak it with a method that will give good isolation of colonies. Use the original broth culture for streaking.

         1.3.1.1.1.1, Incubate for 24 hours at the temperature that seemed optimal before (20°c or 37°c)

           1.3.1.1.1.1.1, Incubate the agar plate, inverted, at the presumed best temperature

     1.3.1.2, one nutrient gelatin deep

       1.3.1.2.1, Make a stab inoculation into the gelatin deep by stabbing the inoculating needle (straight wire) directly down into the medium to the bottom of the tube and pulling it straight out. The medium must not be disturbed laterally.

         1.3.1.2.1.1, Incubate for 24 hours at the temperature that seemed optimal before (20°c or 37°c)

     1.3.1.3, two nutrient broths (The reason for inoculating two tubes of nutrient broth here is to recheck the optimum growth temperature of your unknown.

You incubated your nutrient agar slants at 20?C and 37?C. It may well be that the optimum growth temperature is closer to 30?C. It is to check out this intermediate temperature that an extra nutrient broth is being inoculated.)

       1.3.1.3.1, Inoculate the tubes of nutrient broth with a loop.

         1.3.1.3.1.1, Incubate one of them for 24 hours at the temperature that seemed optimal before(20°c or 37°c); Incubate the remaining tube of nutrient broth separately at 30°c.

     1.3.1.4, one tube of fluid thioglycollate medium

       1.3.1.4.1, Inoculate the tube of FTM with a loopful of your unknown. Mix the organisms throughout the tube by rolling the tube between your palms(laminar hood shakers or mixers are used as the same and more reliable)

         1.3.1.4.1.1, Incubate for 24 hours at the temperature that seemed optimal before (20°c or 37°c)

   1.3.2, Second period (Evaluation) (After the cultures have been properly incubated, carry them to your desk in a careful manner to avoid disturbing the growth pattern in the nutrient broths and FTM)

     1.3.2.1, Before studying any of the tubes or plates, place the tube of nutrient gelatin in an ice water bath

       1.3.2.1.1, Check the Nutrient Agar Slant (Reserve Stock)

         1.3.2.1.1.1, Compare your results with the Nutrient Agar Slant (Reserve Stock): Examine your reserve stock agar slant of your unknown that has been stored in the refrigerator since the last laboratory period. Evaluate it in terms of the following criteria

           1.3.2.1.1.1.1, Amount of Growth

             1.3.2.1.1.1.1.1, The abundance of growth may be described as none, slight, moderate, and abundant

           1.3.2.1.1.1.2, Color

             1.3.2.1.1.1.2.1, Pigments can be associated with a colony, for example, prodigiosin, the red pigment made by Serratia marcescens when grown at 27?C. However, pigments can be produced by an organism that diffuses into the medium, causing the medium to be colored, such as the case for the green fluorescent pigment produced by Pseudomonas fluorescens. To check for diffusable pigments, hold your plate up to the light and observe the color of the medium in the plate. Most bacteria, however, do not produce pigments, and their colonies are white or buff colored

           1.3.2.1.1.1.3, Opacity

             1.3.2.1.1.1.3.1, Organisms that grow prolifically on the surface of a medium will appear more opaque than those that exhibit a small amount of growth. Degrees of opacity may be expressed in terms of opaque, transparent, and translucent (partially transparent)

           1.3.2.1.1.1.4, Form

             1.3.2.1.1.1.4.1, The gross appearance of different types of growth

               1.3.2.1.1.1.4.1.1, Filiform: characterized by uniform growth along the line of inoculation

               1.3.2.1.1.1.4.1.2, Echinulate: margins of growth exhibit toothed appearance

               1.3.2.1.1.1.4.1.3, Beaded: separate or semiconfluent colonies along the line of inoculation

               1.3.2.1.1.1.4.1.4, Effuse: growth is thin, veil-like, unusually spreading 

               1.3.2.1.1.1.4.1.5, Arborescent: branched, treelike growth

               1.3.2.1.1.1.4.1.6, Rhizoid: rootlike appearance

       1.3.2.1.2, Nutrient Broth

         1.3.2.1.2.1, The nature of growth on the surface, subsurface, and bottom of the tube is significant in nutrient broth cultures. Describe your cultures as thoroughly as possible on the descriptive chart with respect to these characteristics

           1.3.2.1.2.1.1, Surface

             1.3.2.1.2.1.1.1, A pellicle type of surface differs from the membranous type in that the latter is much thinner. A flocculent surface is made up of floating adherent masses of bacteria

           1.3.2.1.2.1.2, Subsurface

             1.3.2.1.2.1.2.1, Below the surface, the broth may be described as turbid if it is cloudy, granular if specific small particles can be seen, flocculent if small masses are floating around, and flaky if large particles are in suspension

           1.3.2.1.2.1.3, Sediment

             1.3.2.1.2.1.3.1, The amount of sediment in the bottom of the tube may vary from none to a great deal. To describe the type of sediment, agitate the tube, putting the material in suspension. The type of sediment can be described as granular, flocculent, flaky, and viscid. Test for viscosity by probing the bottom of the tube with a sterile inoculating loop

           1.3.2.1.2.1.4, Amount of Growth

             1.3.2.1.2.1.4.1, To determine the amount of

growth, it is necessary to shake the tube to disperse the organisms. Terms such as slight, moderate, and abundant adequately describe the amount

           1.3.2.1.2.1.5, Temperature Requirements

             1.3.2.1.2.1.5.1, To determine which temperature produces better growth, transfer the contents of the nutrient broth tubes to separate cuvettes and measure the optical density (absorbance) with a spectrophotometer. Because the cultures may be too turbid to measure, you may have to dilute the cultures with water before taking the readings. Record in the descriptive chart which temperature produces better growth for your organism. This temperature will be closer to the one needed for optimum growth of your organism

       1.3.2.1.3, Fluid Thioglycollate Medium

         1.3.2.1.3.1, The growth pattern of your bacterium in fluid thioglycollate medium will give some indication of the oxygen requirement of your organism. Examine your FTM tube and compare the growth pattern of your organism with that of figure 24.5(that mentioned here). More than likely, your bacterium will be either aerobic, microaerophilic, or a facultative anaerobe. Strict anaerobes such as Clostridium require special culture conditions for growth

       1.3.2.1.4, Gelatin Stab (Some bacteria produce proteases, enzymes that degrade proteins. Determine if your unknown produces proteases by examining the nutrient gelatin tube that you inoculated with your unknown)

         1.3.2.1.4.1, After incubation, place the culture in an ice bath and allow it to stand for several minutes. Remove the tube and tilt it several times from side to side to ascertain if liquefaction has occurred. Any degraded gelatin will remain liquid after being placed in the ice bath. If liquefaction has not occurred, the contents of the tube will be a solid. Also be sure to note if your organism can grow in gelatin since some bacteria are unable to do so. Check the configuration with figure that given, to see if any of the illustrations match your tube. A description of each type follows

           1.3.2.1.4.1.1, Liquefaction

             1.3.2.1.4.1.1.1, Crateriform

               1.3.2.1.4.1.1.1.1, saucer-shaped liquefaction

             1.3.2.1.4.1.1.2, Napiform

               1.3.2.1.4.1.1.2.1, turnip-like

             1.3.2.1.4.1.1.3, Infundibular

               1.3.2.1.4.1.1.3.1, funnel-like or inverted cone

             1.3.2.1.4.1.1.4, Saccate

               1.3.2.1.4.1.1.4.1, elongate sac, tubular, cylindrical

             1.3.2.1.4.1.1.5, Stratiform

               1.3.2.1.4.1.1.5.1, liquefied to the walls of the tube in the upper region

           1.3.2.1.4.1.2, No Liquefaction

             1.3.2.1.4.1.2.1, If no liquefaction has occurred, check the tube to see if the organism grows in nutrient gelatin (some do, some don’t). If growth has occurred, compare the growth with the top of the illustration in figure 35.3. It should be pointed out, however, that, from an identification standpoint, the nature of growth in gelatin is not very important

         1.3.2.1.4.2, Note: The configuration of liquefaction is not as significant as the mere fact that liquefaction takes place. If your organism liquefies gelatin, but you are unable to determine the exact configuration, don’t worry about it. However, be sure to record on the descriptive chart the presence or absence of protease production. 

         1.3.2.1.4.3, Another important point: Some organisms produce protease at a very slow rate. Tubes that are negative should be incubated for another 4 or 5 days to see if protease is produced slowly.

       1.3.2.1.5, Nutrient Agar Plate

         1.3.2.1.5.1, Colonies grown on plates of nutrient agar should be

studied with respect to size, color, opacity, form, elevation, and margin. With a dissecting microscope or hand lens, study individual colonies carefully. Refer to figure that is given, for descriptive terminology. Record your observations on the descriptive chart

 1.4, physiological (biochemical) characteristics

   1.4.1, First steps (O/F test, MRVP, CATALASE, OXIDASE & NITRATE REDUCTION)

     1.4.1.1, General information 

       1.4.1.1.1, Keep in mind that it is not routine practice to perform all the tests in identifying an unknown. The “shotgun” method of using all the tests is to be avoided because it is wasteful and can lead to confusing results.

         1.4.1.1.1.1, fermentation tests

           1.4.1.1.1.1.1, O/F glucose

             1.4.1.1.1.1.1.1, If the O/F glucose test determines that your organism is oxidative and not capable of fermenting sugars, then your bacterium cannot be identified by fermentation tests, and you will have to rely on other tests to identify your unknown

           1.4.1.1.1.1.2, specific sugar fermentations

           1.4.1.1.1.1.3, mixed-acid fermentation (methyl red [MR] test)

           1.4.1.1.1.1.4, butanediol fermentation (Voges- Proskauer [VP] test)

           1.4.1.1.1.1.5, citrate test 

         1.4.1.1.1.2, oxidative tests

           1.4.1.1.1.2.1, oxidase

           1.4.1.1.1.2.2, catalase

             1.4.1.1.1.2.2.1, Using H2O2 3%

           1.4.1.1.1.2.3, nitrate tests

     1.4.1.2, Procedures 

       1.4.1.2.1, First period(remember to use Control Positive for each one, e.g. E.coli for glucose O/F test)

         1.4.1.2.1.1, Oxidation and Fermentation Tests

           1.4.1.2.1.1.1, O/F glucose

             1.4.1.2.1.1.1.1, Each unknown organism and each test organism will be inoculated into two tubes of O/F glucose by stabbing with an inoculating needle

             1.4.1.2.1.1.1.2, To one of the tubes for each organism, aseptically deliver about 1 ml of sterile mineral oil after you have inoculated the tube. The mineral oil will establish anaerobic conditions in the tube. The tube without the mineral oil will be aerobic; therefore, be sure to loosen the cap about a quarter of a turn to allow access to the air

             1.4.1.2.1.1.1.3, Incubate the tubes at 37?C for 24 hours

             1.4.1.2.1.1.1.4, Record the results and compare them to the data in table given before here, and the results in figure given here

             1.4.1.2.1.1.1.5, Note: If your tubes do not show any color change from the uninoculated control at 24 hours, incubate them for an additional 48 hours and read them again

         1.4.1.2.1.2, (O/F Durham sugar tubes) Lactose & mannitol O/F tests as the most important ones, both aerobically and anaerobically (if required), method is the same as O/F glucose 

           1.4.1.2.1.2.1, Incubate the carbohydrate tubes, the Simmon’s citrate tube, and the TSA plate for 24 hours

           1.4.1.2.1.2.2, Note: Positive gas production should only be recorded when at least 10% of the medium has been

displaced from the Durham tube

           1.4.1.2.1.2.3, NOTE: Each sugar broth is supplemented with a specific carbohydrate at a concentration of 0.5% as well as beef extract or peptone to satisfy the nitrogen requirements of most bacteria. It is reasonable to assume that your unknown may ferment other sugars, but glucose, lactose, and mannitol are reasonable choices to start with as they are important in differentiating some of the medically important bacteria

         1.4.1.2.1.3, Label one-half of a TSA plate with your unknown and the other half with Pseudomonas aeruginosa. This plate will be used in the next section for oxidative tests to determine if your organism produces the respiratory enzyme cytochrome oxidase

           1.4.1.2.1.3.1, Incubate the carbohydrate tubes, the Simmon’s citrate tube, and the TSA plate for 24 hours

         1.4.1.2.1.4, Inoculate the MR-VP broth tubes with your unknown

           1.4.1.2.1.4.1, Incubate the MR-VP broth tubes for 3–5 days

         1.4.1.2.1.5, Using an inoculating needle, first inoculate the Simmon’s citrate slant by streaking the slant, and then stab the center of the slant about threequarters of the way down into the butt of the tube

           1.4.1.2.1.5.1, Incubate the carbohydrate tubes, the Simmon’s citrate tube, and the TSA plate for 24 hours

       1.4.1.2.2, Second period (Interpretation of the Results) (Test Evaluations)

After 24 to 49 hours’ incubation, arrange all your tubes with the unknown tubes in one row and the test controls in another. As you interpret the results, record the information in the descriptive chart on page 245. Do not trust your memory. Any result that is not properly recorded will have to be repeated.)

         1.4.1.2.2.1, Carbohydrates in Durham Tubes

           1.4.1.2.2.1.1, Concepts 

             1.4.1.2.2.1.1.1, If an organism ferments a sugar, acid is usually produced, and gas may also be an end product of the fermentation. The presence of acid is indicated by a color change in the pH indicator, phenol red, from red at alkaline pH values to yellow at acidic pH values. The production of gas such as hydrogen and carbon dioxide is revealed by the displacement of medium from the Durham tube

               1.4.1.2.2.1.1.1.1, NOTE: Each sugar broth is supplemented with a specific carbohydrate at a concentration of 0.5% as well as beef extract or peptone to satisfy the nitrogen requirements of most bacteria. It is reasonable to assume that your unknown may ferment other sugars, but glucose, lactose, and mannitol are reasonable choices to start with as they are important in differentiating some of the medically important bacteria

               1.4.1.2.2.1.1.1.2, Note: Positive gas production should only be recorded when at least 10% of the medium has been displaced from the Durham tube

           1.4.1.2.2.1.2, Interpretation of the Results

             1.4.1.2.2.1.2.1, Examine the glucose tube inoculated with E. coli.

Note that the phenol red has turned from red to yellow, indicating the presence of acids from the fermentation of the glucose. Also note if medium has been displaced from the Durham tube. If at least 10% of the liquid has been displaced, it means that gas has been formed from the fermentation of the sugar

             1.4.1.2.2.1.2.2, Now examine the test tubes with the test sugars, glucose, lactose, and mannitol that you inoculated with your unknown organism. Record the results for acid and gas production, comparing them to the positive control tubes

         1.4.1.2.2.2, Mixed-Acid Fermentation (Methyl Red Test)

           1.4.1.2.2.2.1, Concepts 

             1.4.1.2.2.2.1.1, An important test in differentiating some of the gramnegative intestinal bacteria is that of mixed-acid fermentation. Genera such as Escherichia, Salmonella, Proteus, and Aeromonas ferment glucose to produce a number of organic acids such as lactic, acetic, succinic, and formic acids. In addition CO2, H2, and ethanol are also produced in this fermentation. To test for the presence of these acids, the pH

indicator, methyl red, is added to the medium, which turns red if acid is present.

           1.4.1.2.2.2.2, Test Procedure

             1.4.1.2.2.2.2.1, Perform the methyl red test first on the control, E. coli, and then on your unknown

         1.4.1.2.2.3, 2,3-Butanediol Fermentation (Voges-Proskauer Test) (The neutral end product, 2,3-butanediol, is not detected directly but must be converted to acetoin by oxidation of the 2,3-butanediol. The acetoin reacts with Barritt’s reagent, which consists of alpha-naphthol and KOH)

           1.4.1.2.2.3.1, Concepts 

             1.4.1.2.2.3.1.1, Some of the gram-negative intestinal bacteria do not carry out mixed-acid fermentation, but rather they ferment glucose to produce limited amounts of some organic acids and primarily a more neutral end product, 2,3-butanediol

             1.4.1.2.2.3.1.2, If an organism produces butanediol and is positive for the VogesProskauer (VP) test, it is usually negative for the methyl red test. The methyl red test and the VogesProskauer test are important tests for differentiating the gram-negative bacteria

             1.4.1.2.2.3.1.3, The reagent is added to a 3- to 5-day old culture grown in MR-VP medium (VP test needs 3-5 days old cultured bacteria) 

             1.4.1.2.2.3.1.4, After adding reagent, vigorously shaken to oxidize the 2,3-butanediol to acetoin. The tube is allowed to stand at room temperature for 30 minutes, during which time the tube will turn pink to red if acetoin is present

           1.4.1.2.2.3.2, Test procedure 

             1.4.1.2.2.3.2.1, Test Procedure Perform the VP test on the control MR-VP broth tube inoculated with Enterobacter aerogenes and on the second MR-VP broth tube inoculated with your unknown organism

               1.4.1.2.2.3.2.1.1, Pipette 1 ml of culture from your unknown to its respective tube and 1 ml of E. aerogenes to its respective tube. Use separate pipettes for each transfer

               1.4.1.2.2.3.2.1.2, Add 18 drops (about 0.5 ml) of Barritt’s reagent A (alpha-naphthol) to each of the tubes containing 1 ml of culture

               1.4.1.2.2.3.2.1.3, Add 18 drops (0.5 ml) of Barritt’s reagent B (KOH) to each of the test tubes

               1.4.1.2.2.3.2.1.4, Cap or cover the mouth of each test tube and shake the tubes vigorously. Allow the tubes to stand for 30 minutes. In this time, the tube with E. aerogenes should turn pink to red. Compare this to your unknown. Vigorous shaking is necessary to oxidize the 2,3-butanediol to acetoin, which reacts with Barritt’s reagents to give the red color

         1.4.1.2.2.4, Oxidase Test

           1.4.1.2.2.4.1, Concepts 

             1.4.1.2.2.4.1.1, The oxidase test assays for the presence of cytochrome oxidase, an enzyme in the electron transport chain. This enzyme catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen, producing oxidized cytochrome c and water (Cytochrome oxidase occurs in bacteria that carry out respiration where oxygen is the terminal electron acceptor; hence, the test differentiates between those bacteria that have cytochrome oxidase and use oxygen as a terminal electron acceptor from those that can use oxygen as a terminal electron acceptor but have other types of terminal oxidases. The enzyme is detected by the use of an artificial electron acceptor, N,N,N?,N?-tetramethyl-p-phenylenediamine, which changes from yellow to purple when electrons are transferred from reduced cytochrome c to the artificial acceptor.

The oxidase test will differentiate most species of oxidase-positive Pseudomonas from the Enterobacteriaceae, which are oxidase negative)

           1.4.1.2.2.4.2, Test Procedure 

             1.4.1.2.2.4.2.1, First method

               1.4.1.2.2.4.2.1.1, Grasp an ampule of oxidase reagent between your thumb and forefinger. Hold the ampule so that it is pointed away from you and squeeze the ampule until the glass breaks. Tap the ampule gently on the tabletop several times

               1.4.1.2.2.4.2.1.2, Touch a sterile swab to the growth of Pseudomonas aeruginosa on the TSA plate. Deliver several drops of oxidase reagent to the cells on the swab. (Note: You do not have to remove the cap of the oxidase reagent as it has a small hole for delivery of the reagent.)

             1.4.1.2.2.4.2.2, Alternative method (easier method); don't forget to check with positive control 

               1.4.1.2.2.4.2.2.1, Transfer growth from the TSA plate to a piece of filter paper and add several drops of reagent to the cells on the paper

               1.4.1.2.2.4.2.2.2, A positive culture will cause the reagent to turn from yellow to purple in 10 to 30 seconds. A change after 30 seconds is considered a negative reaction

               1.4.1.2.2.4.2.2.3, Repeat the test procedure for your unknown organism and record the results in the descriptive chart

         1.4.1.2.2.5, Catalase Test

           1.4.1.2.2.5.1, Concepts 

             1.4.1.2.2.5.1.1, When aerobic bacteria grow by respiration, they use oxygen as a terminal electron acceptor, converting it to water. However, they also produce hydrogen peroxide as a by-product of this reaction. Hydrogen peroxide is a highly reactive oxidizing agent that can damage enzymes, nucleic acids, and other essential molecules in the bacterial cell. To avoid this damage, aerobes produce the enzyme catalase, which degrades hydrogen peroxide into harmless oxygen and water

           1.4.1.2.2.5.2, Test procedure

             1.4.1.2.2.5.2.1, To determine if catalase is produced, a small amount of growth is transferred from a plate or slant, using a wooden stick, to a clean microscope slide. A couple of drops of 3% hydrogen peroxide are added to the cells on the slide. If catalase is produced, there will be vigorous bubbling due to the breakdown of hydrogen peroxide and the production of oxygen gas

               1.4.1.2.2.5.2.1.1, Note: Do not use a wire loop to transfer and mix the cells as iron can cause the hydrogen peroxide to break down, releasing oxygen. Also, do not perform the catalase test on cells growing on blood agar since blood contains catalase

         1.4.1.2.2.6, Nitrate Reduction

           1.4.1.2.2.6.1, Concepts 

             1.4.1.2.2.6.1.1, Some facultative anaerobes can use nitrate as a terminal electron acceptor in a type of anaerobic respiration called nitrate respiration. Bacteria such as Paracoccus and some Pseudomonas and Bacillus reduce nitrate to a gaseous end product such as N2O or N2. Other bacteria such as Escherichia coli partially reduce nitrate to nitrite. Several enzymes are involved in the reduction of nitrate, one of which is nitrate reductase, which catalyzes the transfer of electrons from cytochrome b to nitrate, reducing it to nitrite

             1.4.1.2.2.6.1.2, Cultures are grown in beef extract medium containing potassium nitrate. Gases produced from nitrate reduction are captured in Durham tubes placed in the nitrate medium. Partial reduction of nitrate to nitrite is assayed for by adding sulfanilic acid (reagent A) followed by dimethyl-alpha-naphthylamine (reagent B). If nitrite is produced by reduction, it will form a chemical complex with the sulfanilic acid and the dimethylalpha-naphthylamine to give a dark red color

               1.4.1.2.2.6.1.2.1, A negative test could mean that nitrate was not reduced or that some other reduced form of nitrogen was produced, such as ammonia or hydroxlyamine. As a check, zinc powder is added to the test medium. Zinc metal will chemically reduce nitrate to nitrite, causing the medium to turn dark red as result of the formation of the chemical complex. If a nongaseous product such as ammonia was produced, no color will develop after the addition of the zinc metal

           1.4.1.2.2.6.2, Test procedure 

             1.4.1.2.2.6.2.1, Examine the nitrate broth of your unknown. If gas has been displaced in the Durham tube, it means that your organism has reduced nitrate to a gaseous end product, such as nitrogen gas. If no gas is present, reduction may have resulted in the formation of nitrite or the formation of a nongaseous end product

             1.4.1.2.2.6.2.2, To test for the presence of nitrite, first assay the test control E. coli culture by adding 2 to 3 drops of reagent A and 2 to 3 drops of reagent B to the nitrate broth culture of the organism. A deep red color will develop immediately

             1.4.1.2.2.6.2.3, Caution Avoid skin contact with solution B. Dimethyl-alphanaphthylamine is carcinogenic

             1.4.1.2.2.6.2.4, Repeat this same test procedure for your unknown bacterium. If a red color fails to appear, your organism did not reduce nitrate or it may have produced a nongaseous end product of nitrate reduction

             1.4.1.2.2.6.2.5, Zinc Test: To the negative culture, add a pinch of zinc powder and shake the tube vigorously. If a red color develops in the tube, nitrate was reduced by the zinc metal, indicating a negative test for nitrate reduction. If no color develops, a nongaseous end product may have been formed, which means your unknown reduced nitrate

   1.4.2, Second steps ( Hydrolytic and Degradative Reactions)

     1.4.2.1, Starch agar

       1.4.2.1.1, Note that straight–line streaks are used on the plates and that test control organisms and unknown organisms are inoculated on the same plate for comparison

         1.4.2.1.1.1, Incubation:

The plates and the tube media should be incubated at the optimum temperature determined for your unknown for 48 hours

           1.4.2.1.1.1.1, Starch hydrolysis: Add several drops of Gram's iodine to the growth on the plate. Blue areas indicate the presence of starch, and clear areas adjacent to growth streaks indicate starch hydrolysis

             1.4.2.1.1.1.1.1, Control Positive: B. subtilis; using straight–line streaks with unknown in the same plate

               1.4.2.1.1.1.1.1.1, Starch hydrolysis is detected by adding Gram's iodine to starch medium. Iodine complexes with the starch macromolecule and causes the medium to turn blue. However, if the starch has been degraded, the medium adjacent to the bacterial growth will be clear after the addition of the iodine

     1.4.2.2, Skim milk agar

       1.4.2.2.1, Note that straight–line streaks are used on the plates and that test control organisms and unknown organisms are inoculated on the same plate for comparison

         1.4.2.2.1.1, Incubation:

The plates and the tube media should be incubated at the optimum temperature determined for your unknown for 48 hours

           1.4.2.2.1.1.1, Casein hydrolysis: Casein has been hydrolyzed if clear areas are seen adjacent to growth

             1.4.2.2.1.1.1.1, Control Positive: B. subtilis ; using straight–line streaks with unknown in the same plate

               1.4.2.2.1.1.1.1.1, Examine the growth on the skim milk agar. Note

the clear zone surrounding the bacterium in the streak, which illustrates casein hydrolysis. Compare with your unknown

     1.4.2.3, Spirit blue agar

       1.4.2.3.1, Note that straight–line streaks are used on the plates and that test control organisms and unknown organisms are inoculated on the same plate for comparison

         1.4.2.3.1.1, Incubation:

The plates and the tube media should be incubated at the optimum temperature determined for your unknown for 48 hours

           1.4.2.3.1.1.1, Fat hydrolysis: If growth streak exhibits a dark blue precipitate, the organism is positive for fat hydrolysis

             1.4.2.3.1.1.1.1, Control Positive: S. aureus ; using straight–line streaks with unknown in the same plate

               1.4.2.3.1.1.1.1.1, Spirit blue agar contains peptone as a source of carbon, nitrogen, and vitamins. It also contains tributyrin, a simple, natural animal triglyceride that serves as a substrate for lipases. Release of the fatty acids from tributyrin via lipase activity results in the lowering of the pH of the agar to produce a dark blue precipitate. However, some bacteria do not completely hydrolyze all the fatty acids from the tributyrin, and as a result, the pH is not sufficiently lowered to give the dark blue precipitate. In this case, all you notice may be simply the depletion of fat or oil droplets in the agar to indicate lipase activity

                 1.4.2.3.1.1.1.1.1.1, Examine the growth of S. aureus on the plate. You should be able to see the dark blue reaction. Compare this to your unknown. If your unknown appears negative, hold the plate up toward the light and look for a region near the growth where oil droplets are depleted. If you see the depletion of oil droplets, record this as a positive test in the descriptive chart

     1.4.2.4, Tryptone broth

       1.4.2.4.1, Incubation:

The plates and the tube media should be incubated at the optimum temperature determined for your unknown for 48 hours

         1.4.2.4.1.1, Tryptophan hydrolysis: Add Kovac’s reagent to broth tube. Development of a red ring on the surface of the broth indicates that indole has been produced from the hydrolysis of tryptophan

           1.4.2.4.1.1.1, Test control tube inoculations: A tryptone broth (indole production) is inoculated with E. coli; Incubate at 37°C for 24 to 48 hours

             1.4.2.4.1.1.1.1, The enzyme responsible for the cleavage of tryptophan is tryptophanase. The degradation of tryptophan by the enzyme can be detected with Kovac’s reagent, which forms a deep red color if indole is present. Tryptone broth (1%) is used for the test because it contains high amounts of tryptophan

               1.4.2.4.1.1.1.1.1, To test for indole and therefore the activity of tryptophanase, add 10 to 12 drops of Kovac’s reagent to the tryptone broth culture of E. coli. A red organic

layer should form on top of the culture. Repeat the test for your unknown culture and record the results on the descriptive chart

     1.4.2.5, Urea agar

       1.4.2.5.1, Incubation:

The plates and the tube media should be incubated at the optimum temperature determined for your unknown for 48 hours

         1.4.2.5.1.1, Urea hydrolysis: If the agar slant exhibits a cerise color, the organism can hydrolyze urea

           1.4.2.5.1.1.1, Test control tube inoculation: A urea agar slant are inoculated with P. vulgaris. Incubate at 37°C for 24 to 48 hours

             1.4.2.5.1.1.1.1, Urea is a waste product of animal metabolism that is broken down by a number of bacteria. The enzyme responsible for urea hydrolysis is urease, which splits the molecule into carbon dioxide and ammonia. Urea medium contains yeast extract, urea, a buffer, and the pH indicator phenol red. Urea is unstable and is broken down by heating under steam pressure at 15 psi. Therefore, the medium is prepared by adding filtersterilized urea to the base medium after autoclaving it.

               1.4.2.5.1.1.1.1.1, When urease is produced by an organism, the resulting ammonia causes the pH to become alkaline. As the pH increases, the phenol red changes from yellow (pH 6.8) to a bright pink or cerise color (pH 8.1 or greater)

                 1.4.2.5.1.1.1.1.1.1, Examine the urea slant inoculated with Proteus vulgaris and compare it to your unknown. If your urea slant is negative, continue the incubation for an additional 7 days to check for slow urease production. Record your results in the descriptive chart

     1.4.2.6, Phenylalanine agar (Phenylalanine Deamination - PPA test)

       1.4.2.6.1, Incubation:

The plates and the tube media should be incubated at the optimum temperature determined for your unknown for 48 hours

         1.4.2.6.1.1, Phenylalanine deamination: Allow 5–10 drops of 10% ferric chloride to flow down the surface of the slant. Mix with an inoculationg loop. If phenylalanine has been deaminated, a deep green color will appear after 1–5 minutes

           1.4.2.6.1.1.1, Test control tube inoculation: A phenylalanine agar slant are inoculated with P. vulgaris. Incubate at 37°C for 24 to 48 hours

             1.4.2.6.1.1.1.1, Gram-negative bacteria such as Proteus, Morganella, and Providencia can oxidatively deaminate the amino acid phenylalanine to produce phenylpyruvic acid and ammonia. The reaction is catalyzed by the enzyme phenylalanine deaminase, a flavoprotein oxidase. The enzyme can be detected by the addition of 10% ferric chloride, which forms a green-colored complex with ?-keto acids such as phenylpyruvic acid. The test is useful in differentiating the above bacteria from other Enterobacteriaceae.

               1.4.2.6.1.1.1.1.1, Allow 5 to 10 drops of 10% ferric chloride to

flow down the slant of the test control organism, P. vulgaris. To facilitate the reaction, use an inoculating loop to emulsify the culture on the slant with the test reagent. A deep green color should appear in 1 to 5 minutes. Repeat the test procedure for your unknown. Record your results in the descriptive chart

   1.4.3, Multiple Test Media

     1.4.3.1, Kligler’s Iron Agar or TSI (test evaluation)

       1.4.3.1.1, 1. Alkaline (red) slant/acid (yellow) butt (figure 38.1a): This means that only glucose was utilized. The organism utilized the low concentration of glucose initially and then degraded the peptone in the medium. The slant is alkaline (red) because glucose was degraded aerobically, and the ammonia released from peptone utilization caused the aerobic slant to become alkaline. However, the butt is yellow (acid) because glucose was fermented anaerobically to produce enough acids to cause the acidic reaction in the butt. If gas is produced, it will be evident by the splitting of the medium and the formation of gas bubbles in the agar slant

       1.4.3.1.2, 2. Acid (yellow) slant/acid (yellow) butt: The organism has fermented both glucose and lactose, producing acids that cause the pH indicator to turn yellow. Lactose is present in 10 times (1%) the concentration of glucose (0.1%), and sufficient acid is produced to cause both the slant and butt to be acidic. However, the tubes must be read at 24 hours because they can revert to alkaline in 48 hours if the lactose becomes depleted and the peptones are utilized, producing ammonia

       1.4.3.1.3, 3. Alkaline (red) slant/alkaline (red) butt; alkaline (red) slant/no change butt: No fermentation of either sugar has occurred. Some enteric bacteria can use the peptones both aerobically and anaerobically, causing both the slant and butt to become alkaline. Others can only use the peptone aerobically, producing an alkaline slant but no change in the butt

       1.4.3.1.4, These cultures contains ferrous salts that will react with the hydrogen sulfide liberated by cysteine desulfurase to produce an insoluble black precipitate

     1.4.3.2, SIM Medium

       1.4.3.2.1, SIM medium is a multiple test medium that detects the production of hydrogen sulfide and indole and determines if an organism is motile or not. The medium contains hydrolyzed casein, ferrous salts, and agar (0.7%), which makes the medium semisolid. It is inoculated by stabbing. The breakdown of tryptophan in the medium will produce indole, which can be detected by adding Kovac’s reagent. If cysteine is degraded, hydrogen sulfide will be released, which will combine with the ferrous salts to produce a black precipitate in the tube

     1.4.3.3, The IMViC Tests

       1.4.3.3.1, If your organism is a gram-negative rod and a facultative anaerobe, group these tests and see how your organism fits the combination of tests

     1.4.3.4, Litmus Milk

       1.4.3.4.1, Acid, no clot

         1.4.3.4.1.1, Fermentation of lactose and/or dextrose produces acids that cause the litmus to turn pink

       1.4.3.4.2, Alkaline

         1.4.3.4.2.1, Breakdown of milk proteins such as lactalbumin results in the release of ammonia and amines that cause an alkaline reaction and blue color

       1.4.3.4.3, Peptonization

         1.4.3.4.3.1, Digestion of milk proteins is evidenced by a clearing of the medium

       1.4.3.4.4, Reduction

         1.4.3.4.4.1, Reductase enzymes cause the removal of oxygen and the decolorization of the litmus

       1.4.3.4.5, Stormy Fermentation and Coagulation

         1.4.3.4.5.1, k proteins resulting in the formation of a curd or clot

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Hope it could be useful for anyone who may like to fill the gap of what may never told in practices...

all of these information shared for students (as my friend!) to evaluate and understand what may told them to do without understanding the methods and how to isolate logically their target bacteria from different microbiome of soil and water; Gelatin agar is choice for finding proteases & Skim milk agar for finding caseinase! please don't mislead your students! Thank you...