Biochemical Activities

Biochemical Activities
By: Amanda Goldner

Table of Contents

Lab A: Media Preparation
Lab 1: Inoculations, Smear Preps, and Simple Staining
Lab 2: Gram Staining and Using the Spectrophotometer
Lab 3: Negative, Acid Fast, Endospore, and Capsule Staining
Lab 4: Biochemical Activities of Bacteria I ……………………………………….… 3

Lab 5: Bergey’s Manual (Practical) …………………………………………………  11
Lab 6: Biochemical Activities of Bacteria II, Unknown Identification, and
Rapid Multitests …………………………………………………………………………………………… 13
Lab 7: Isolation of Microorganisms From Soil
Lab 8: Eukaryotes I: Fungi
Lab 9: Eukaryotes II: Algae and Protozoa
Lab 10: Poster Presentations

Lab 4: Biochemical Activities of Bacteria I
I. Purpose

The objective of this lab was to run differential tests on Escherichia coli, Pseudomonas fluorescens, Bacillus subtilis, and Proteus vulgaris. This was done to determine such characteristics as ability to ferment lactose/glucose/sucrose, produce hydrogen sulfide/acids/acetoin, and utilize urea or citrate.

II. Materials
The materials needed for this lab were as follows:

– inoculating loop
– disposable gloves
– inoculating needle
– permanent marker
– Bunsen burner
– 1 Eosin Methylene Blue (EMB) agar plate
– 1 Hektoen agar plate
– 1 McConkey agar plate
– Barritts A reagent
– Barritts B reagent
– Kovac’s reagent
– biohazard container
– 4 Pasteur pipettes
– 4 Sulfide-Indole Motility (SIM) agar deeps
– 4 Triple Sugar Iron (TSI) agar slants
– 4 urea agar slants
– 4 Simmons Citrate Agar (SCA) slants
– 4 Glucose Phosphate Broth tubes
– 4 empty test tubes
– tryptic soy agar plate cultures (pure) of:

~ Escherichia coli

~ Bacillus subtilis

~ Pseudomonas fluorescens
~ Proteus vulgaris

III. Methods

McConkey, EMB, and Hektoen Agar Plates

    The McConkey, Eosin Methylene Blue (EMB), and Hektoen plates were each inoculated with a streak of every bacterial variety listed in the Materials section of this report. Each plate had four resulting one-inch streaks, one in each quadrant and each made up of a different species inoculum. Aseptic technique was maintained in all bacterial transfers (ex:  flaming the inoculating loop at proper times).  The plates were labeled on the bottom with permanent marker, split into four quadrants for each streak.  The genus species initials for each bacterial type were written down in the respective quadrant in which that species was located.  All three inoculated plates were then placed on a storage rack to be incubated for 24 hours.

Triple Sugar Iron (TSI) Agar Slants

    The four Triple Sugar Iron agar slants were each inoculated via inoculating needle with a different species of bacteria (those listed in Materials section).  Proper aseptic technique concerning inoculation and test tubes was maintained in all bacterial transferals.  The TSI slant was first stabbed with the needle, before the needle was dragged in a zigzag pattern across the surface of the slant from the lower-elevation end to the higher-elevation end.  The needle was not flamed between the stabbing and streaking of the agar.  Once this procedure had been applied to all slants, the tubes were placed in the storage rack for a 24-hour period incubation.

Simmons Citrate (SCA) and Urease Agar Slants

    One Simmons Citrate Agar slant and one urease slant was reserved for each species of bacteria listed in the Materials section above.  All slants were inoculated via inoculation loop in a zigzag pattern across the surface of the slant from the lower-elevation end to the higher-elevation end, like the streak configuration seen in the TSI slants.  Aseptic technique pertaining to test tubes and inoculating instruments was maintained in all bacterial transfers.  However, SCA and urease slants were inoculated with a loop and were not stabbed in the butt.  The freshly-inoculated SCA and urease slants were placed to the side in the storage rack for 24 hours of incubation.

Sulfide-Indole Motility Deeps

    The four Simmons Indole Motility agar deeps were inoculated with a vertical stab from an inoculation needle, each with a different type of bacteria (as listed in the Materials section).  Aseptic technique (such as flaming the inoculating needle at proper times) was maintained in all bacterial transferals.  These new culture deeps were then transferred to the storage rack with the rest of the cultures made previously in the lab to be incubated for 24 hours.

Methyl Red and Voges-Proskauer Broth Preparation

    An inoculating loop was used to transfer an inoculum of each species in the Materials section to its own sterile glucose phosphate broth tube, with aseptic technique being strictly followed regarding tube and loop sterilization.  These broth cultures would later be differentiated into eight MR-VP broths, which would then have MR and VP tests run on them using Barritts A and/or B reagent and methyl red indicator.  The cultures were placed to the side for the moment (in the storage rack) to go through a 24-hour incubation period.

———————————————— COMEBACKS ————————————————-

 

Methyl Red and Voges-Proskauer Tests

    The glucose phosphate broth cultures were removed from incubation; half of each tube’s contents was transferred to a new test tube.  To one 4-species tube set, 4-5 drops of methyl red indicator were added and the test tube was shaken.  These tubes were labeled MR and set aside for a few minutes for observation.  To the other four tubes, 10 drops of Barritts A followed by 10 drops of Barritts B were added.  This set of tubes was shaken every three minutes for 15-20 minutes.  Positive results of the methyl red test indicate a pH of 4; the broths developed a red hue.  Negative results of the MR test turned the indicator yellow; this meant the broth cultures had a pH of 6.  As for the Voges-Proskauer test, positive results (red coloration) indicate the presence of acetoin, while negative results involve absence of red tinge and acetoin.

All result conclusions were denoted onto properly-labeled tables found in the Results section of this report.  These outcomes were then studied (see Discussion) to see if the tests had results consistent with the literature values.

Interpreting Results From Plates

    The McConkey agar plates in this experiment were differential, meaning they discerned between bacteria with dissimilarities in characteristics.  Specifically, McConkey differentiated between lactose fermenters and those bacteria that did not ferment lactose.  Bacteria on McConkey agar had lactose positive results if its colonies turned red or pink after the period of incubation.  Lactose negative results mean white, clear, or golden brown dark-centered colonies.
The Eosin Methylene Blue agar plates used in lab were also differential, determining whether a bacterial species can ferment lactose or not.  Positive results were shown by blue-black coloration (E. coli) or pink coloration (Enterobacter).  Since Enterobacter was not used in this experiment, any resultant pink hues mean nothing, and were classified as lactose negative along with lack of colonial coloration.
The third and final type of plate used in this lab was also categorized as differential, but determined differences in pH instead of fermentation ability.  These “Hektoen” agar plates were originally green in color, and changes in pH effect a striking alteration in color:  orange.  Positive lactose fermentation was indicated by orange colonies and/or medium.  No color change of colonies and medium suggested negative lactose fermentation results.  Hektoen plates also revealed hydrogen sulfide production; if this occurred, colonies would have black centers.  Bacteria without black centers were grouped as H2S negative.
All results were recorded into the corresponding tables in the Results section of this report.  These data were later examined to see if the outcomes were similar to those found in previous experiments.

Interpreting Results From TSI Slants

    The area of the tube referred to as the slant encompassed the surface and a few centimeters below, and was inoculated by the needle streak pattern.  The region termed the “butt” comprised the rest of the slant, and was inoculated by the needle stab line.  TSI slants were initially red in color, due to alkaline pH.  If either the slant or the butt changed to a yellow color, it was an acid positive result.  Alkaline positive outcomes stayed red in color.  As can be assumed, acid negative results meant alkaline positive and vice versa. These acid and alkaline results were separate for each region (slant/butt).  If bubbles or cracks were apparent in the agar, then the culture tested gas positive; absence of bubbles/cracks was considered gas negative.  Presence of black material produced H2S positive results, and absence of said black material meant H2S negative results.

All results were logged in table form in the Results section.  Later analysis, as seen in the Discussion section of this report, told if the tests had literature-consistent outcomes.

Interpreting Results From SCA and Urease Slants

    Simmons citrate agar slants fell into the differential category of media.  Inoculation of these slants with a bacterium established whether that species could utilize citrate or not.  A citrate positive outcome involved a change from the original green color to blue.  Citrate negative results did not affect the color, and remained green.  Presence of growth was also noted for this test, with growth positive implying that growth occurred and growth negative meaning lack of growth.
Urease slants also involved determining growth occurrence, with growth positive and negative results implying the same as those for SCA slants.  Pink coloration signified a positive urease test result (the bacteria utilized urea), while negative results (the bacteria did not utilize urea) lacked deviation from original color.

All outcomes were recorded into the corresponding tables in the Results section for later scrutiny, to see if the tests had results consistent with the literature values.

 

 

Interpreting Results From Sulfide-Indole Motility Deeps

    Seven to ten drops of Kovac’s reagent were added to the four SIM agar deeps after removal from incubation.  Within two minutes, results were observed as follows.  Red coloration on the surface of the tube signified indole positive results; absence of said red hue represented an indole negative end result.
All outcomes were detailed into designated tables (see the Results section).  These data were later inspected to see if the results were correct according to literature values.

IV. Results

 

Broths à

 

Methyl Red

 Voges-Proskauer

growth

acid

growth

acetoin

P. vulgaris

turbid throughout until just below the surface

+

+

E. coli

sedimented

at bottom

+

+

P. fluorescens

layered below surface, none beneath center

+

+

B. subtilis

turbid throughout

+

+

Plates

Hektoen Enteric Agar (HE) Eosin Methylene Blue Agar (EMB) McConkey Agar (MAC)

growth

colony color

lactose fermentation

H2S formation

growth

colony color

lactose fermentation

growth

colony color

lactose fermentation

P. vulgaris

+

orange

+

+

+

E. coli

+

orange

+

+

blue

+

+

red

+

P. fluorescens

+

green

+

+

B. subtilis

+

pink

+

+

red

+

 

Slants

Triple Sugar Iron Agar (TSI)

 

Urease Agar

 

Simmons Citrate Agar (SCA)

Butt

Slant

acid

alkaline

gas

H2S production

acid

alkaline

growth

urea hydrolysis

growth

citrate utilization

P. vulgaris

+

+

+

+

+

+

+

E. coli

+

+

+

+

+

P. fluorescens

+

+

+

+

+

+

B. subtilis

+

+

+

+

 

 

  Deeps

Sulfide-Indole Motility Agar

growth

motility

H2S production

indole

P. vulgaris

+

+

E. coli

+

+

+

P. fluorescens

+

B. subtilis

+

+

V. Discussion
This microbiology lab involved the inoculating of many differential media, including:  EMB plates, MR broths, SCA slants, McConkey plates, TSI slants, SIM deeps, Hektoen plates, urease slants, and VP broths.  Each media had a distinct purpose, some even tested for more than one characteristic.  Results were observed after 24 hours of incubation and are interpreted in the following paragraphs along with explanations of the workings of each media type used in this lab.  Every bacterial species in every type of media test had growth present, except for B. subtilis on Hektoen Enteric Agar, as was discussed later on.
Two words used to describe media in this experiment were “differential” and “selective.” Differential media made a distinction between at least two groups of microorganisms.  Differential media made it possible to narrow down the list of possible identities of an unknown bacterial species by biological characteristics alone.  Meanwhile, selective media discriminated against certain types of microbes and favored others.  These media could have been used to make certain that a bacterial colony was part of a particular group (such as gram positive or gram negative), though this function was not taken advantage of.  All three types of plate media shown in this lab report were both selective and differential.
In this experiment, Eosin Methylene Blue (EMB) agar was used to select for gram negative bacteria, inhibiting the growth of any species that were gram positive.  It also determined whether colonies underwent lactose fermentation.  These lactose fermenters typically changed to a blue or black color, because acid production lowered the pH (increasing methylene blue absorption).  Bacteria under the genus Enterobacter were exceptions, and developed a pink hue after incubation on EMB agar.  Non-fermenting bacteria remained colorless.  EMB agar also allowed for recognition of coliform enteric bacteria.  In this experiment, E. coli and B. subtilis acquired the respective colors indicative of lactose fermentation for their genus (on EMB agar), and thus tested lactose positive.  P. fluorescens and P. vulgaris tested negative for lactose fermentation by appearing colorless, however.
McConkey agar was determined to be selective because it prevented the growth of gram positive bacteria by means of bile salts and crystal violet dye.  The pink coloration of the agar came from the crystal violet.  McConkey plates also included lactose, which was converted to lactic acid by lactose fermentation.  This accumulation of acid effected a change in pH (indicated by Neutral Red), and any colonies that ferment acquired a dark pink-red coloration.  Non-fermenters remain opaque and colorless.  MacConkey plates were also differential because of the ability to detect enteric pathogens and coliforms.  In this lab, E. coli and B. subtilis both has positive results, developing a red coloration that revealed their true nature as lactose fermenters.  P. vulgaris and P. fluorescens remained colorless and were thus not labeled lactose fermenters.
Hektoen Enteric agar (HE, HEM, HEK, or HEA) was the only one out of the three agar plates used in this experiment that could sense hydrogen sulfide (H2S) production; colonies that produced H2S took on a black pigmentation.  The agar itself was comprised of lactose, salicin, and bile, which together gave the medium its selective and differential properties.  Coliform enteric bacteria could be identified by using Hektoen media; these turned orange-pink after 24-48 hours.  Salmonella and Shigella typically became blue-green.  HE, like McConkey and EMB, could also distinguish lactose fermenters and non-lactose-fermenters; the region around the fermenting colonies converted to orange.  Results for lactose fermentation of P. vulgaris came out incorrectly in this experiment; the bacteria appeared to be lactose positive but in reality was lactose negative (this error was probably due to wrongful observations or a mix-up).  B. subtilis did not appear to show any growth at all and thus tested negative for every characteristic distinguished by Hektoen Enteric Agar.  Meanwhile, E. coli and P. fluorescens both seemed to turn out correctly, with development of orange and dark green hues (respectively).  These results implied that E. coli underwent lactose fermentation and P. fluorescens did not.
Triple Sugar Iron agar slants were possibly the most complex form of media used in this lab; incubation for 18-24 hours detected the presence or absence of H2S production, sugar fermentation, and/or gas production. These slants were commonly used by microbiologists to identify enteric bacteria, and contained 1% lactose, 1% sucrose, and 0.1% glucose concentrations.  Originally red in color due to the phenol red indicator, the agar slants were both stabbed and streaked with the inoculating needle.  This method formed two reaction areas in the same tube, one with deoxygenated/anaerobic conditions (the butt) and one with an oxygenated/aerobic environment (the slant).  A partially yellow (acid) butt and red (alkaline) slant meant glucose had been fermented, but coloration due to acid was not widespread because oxidative decarboxylation increases the pH of the slant.  A yellow butt and slant suggested that more than just glucose was fermented, leading to acid production throughout the medium.  An entirely red TSI tube indicated that no sugars had been fermented.  This last result never showed signs of gas production (splitting or bubbles in the agar) or hydrogen sulfide production (one or more black areas).  However, either of these characteristics could have been seen in a TSI tube with some amount of yellow coloration, because both were considered possible side effects of carbohydrate fermentation.  The gas/H2S production in TSI tubes was a byproduct of the reduction of thiosulfate to sulfite.
The Triple Sugar Iron tests seemed to turn out correctly in this lab, despite the many possibilities for human error.  The slant culture of P. vulgaris developed an acid slant and alkaline butt, which implied that a limited amount of carbohydrate fermentation had occurred, resulting in one of two possible byproducts:  hydrogen sulfide production.  E. coli’s slant was entirely acidic and yellow in color, a strong indication of carbohydrate fermentation.  Acid was not the only sign of this process, however, large amounts of bubbles and cracks in the agar lead to the assumption that gas had been produced as a byproduct.  TSI cultures of B. subtilis and P. fluorescens produced the same result:  an entirely alkaline tube without presence of H2S or gas.
The urease test detects for urease activity, or an organism’s ability to utilize urea.     These bacteria (seen in lab as P. vulgaris and P. fluorescens) make an enzyme that attacks the carbon-nitrogen bonds in amide compounds like urea.  This reaction produced CO2, NH3, and H2O, changing the originally straw-colored urease slant to a hot pink (positive test).  This hot pink color came from the pH indicator phenol red’s response to a sudden rise in pH from excess ammonia production.  Negative tests are still straw-colored.  As mentioned before, P. vulgaris and P. fluorescens both tested positive for urea utilization, producing the amide C-N bond-attacking enzyme to effect a swift color change in the urease slant to a bright pink.
Simmons Citrate Agar (SCA) tests determined a bacterial species’ ability to use sodium citrate as its only carbon source to produce energy.  If the species was equipped with a citrate permease to transport citrate into its cells, then the bacteria could convert the citrate to carbon dioxide and pyruvic acid.  This carbon dioxide combined with Na+ from the sodium citrate to produce alkaline sodium carbonate.  SCA tests also included NH4+ as a nitrogen source and bromothymol blue as a pH indicator.  Sodium carbonate production raises the pH and turns bromothymol blue from green to blue, indicating alkalinity.  Since citrate utilization requires oxygen, SCA tests are made in slant form.  Citrate negative tests normally should not have had presence of bacterial growth; however, this seemed to occur on both negative SCA results found in this lab (for E. coli and B. subtilis).  This could have been due to contamination from outside sources during inoculation, or a non-sterile SCA test tube.  P. vulgaris and P. fluorescens, on the other hand, both tested positive for citrate utilization, meaning they both were equipped with a citrate permease and could use sodium citrate as the sole carbon source.
Sulfide-Indole Motility (SIM) tests determined a bacterial colony’s amount of movement, hydrogen sulfide production, and indole production.  Indole was one of the byproducts of tryptophan hydrolysis, along with pyruvic acid and ammonia.  The NH4 and pyruvic acid got used for nutritional purposes by the bacteria, leaving an excess of indole (indicated by a formation of red coloration several minutes after the addition of Kovac’s reagent).

These SIM agar deeps were stabbed with the inoculating needle and incubated overnight, resultant appearances of tubes were examined to produce the following conclusions:  E. coli and B. subtilis both seemed to be motile bacterial species due to their apparent growth away from the line of stab inoculation.  Of the two, E. coli was the only one to reduce the thiosulfate in the tube to H2S, as was seen by the presence of black material in the tube.  P. vulgaris also produced hydrogen sulfide, but did not test positive for motility because the culture’s growth was restricted to the stab line.  According to the literature, however, this was incorrect, as P. vulgaris was a motile species.  The actual observation of PV’s test tube was probably mistaken, as the growth may not have been very far past the line of inoculation but was still enough to count as motile.  P. vulgaris’ indole test (performed by addition of Kovac’s reagent) also came out wrong, a false negative.  The likely cause of this error was that the Kovac’s reagent was not given enough time to react before observations were recorded two minutes later.  E. coli also had a false negative indole test result; meanwhile, the negative outcomes for P. fluorescens and B. subtilis were correct.  Observations of the SIM tubes after incubation lead to further conclusions that B. subtilis was motile but did not produce H2S, while P. fluorescens was neither motile or a thiosulfate reducer.

Methyl red tests were named after the pH indicator they contained, which developed a red hue if acidic end products of glucose catabolism were present.  Mixed acid fermenters acidified the medium by producing a variety of fermentation acids, which caused a greater fall in pH level than the effects of butanediol fermenters (production of butanediol, various organic acids, and acetoin).  Methyl red turns red (positive result) at a pH of 4, and yellow (negative result) at a pH of 6.  P. vulgaris and E. coli both had positive MR test results, with a switch to a red tinge after the addition of 4-5 drops of methyl red.  P. fluorescens and B. subtilis did not change in color once methyl red was added, but remained opaque (negative results).  These outcomes implied that P. vulgaris and E. coli both created acidic end products in their catabolism of glucose, while P. fluorescens and B. subtilis either did not ferment glucose or did not produce the same amounts of acidic products.

VI. References
(Harley JP, 2011). Laboratory Exercises in Microbiology 8th Edition: 139-143, 155-166, 195-197.

Lab 5: Bergey’s Manual

 

I. Purpose

The objective of this lab was to practice using Bergey’s Manual of Systematic Bacteriology to identify an unknown bacterial strain based on its biochemical properties alone.

II. Materials

The materials needed for this lab were as follows:
Bergey’s Manual of Systematic Bacteriology
- question sheet with detailed descriptions of two unknown bacteria

III. Methods

Identification of Unknown Bacteria

Bergey’s Manual of Systematic Bacteriology was skimmed through until a genus with similar or the same biochemical characteristics as one of the unknown bacteria on the question sheet.  The following sections of each article were used to identify the bacteria correctly:  name of genus, capsule description, enrichment, isolation, taxonomic comment(s), differential list of species within the genus, differences between the genus and other genera, and any further descriptive information.  These portions of each section were used to narrow down the possible identities of the unknown bacterial species from infinite to a definite match.  These definite matches (genus species names) were recorded as the answers to the questions on the question sheet.

IV. Results
The question sheet was turned in at the end of the lab, so there were no results to show here.
V. Discussion

Bergey’s Manual of Systematic Bacteriology was a fairly efficient method to identify bacterial unknowns from a comprehensive list of all biochemical characteristics that could be determined using standard differential tests.  There were four volumes in total, but only the ones pertaining to the bacterial species on the question sheet were skimmed through.  Introductory articles on bacterial taxonomy were located at the beginning of each volume for greater speed of identification.  The section of an article mentioned in the Methods section, “further descriptive information,” includes informative reads on topics pertaining to the genus, such as:  physiology, growth conditions, nutrition requirements, genetics, ability to cause disease, metabolism, ecology, and morphology.  These articles on bacterial genera include three main types of tables:  tables that differentiated between the species in the genus, tables that compared the respective genus to related genera, and tables that provided additional information on a particular species in the genus (possibly, this species is more commonly used in microbiology laboratories than others).

Lab 6: Biochemical Activities of Bacteria II, Unknown Identification, and Rapid Multitests
I. Purpose

The objective of this lab was to practice using rapid multitests such as the Enterotube II and API 20E to identify unknown bacterial specimens.  Also, the biochemical activities of various combinations of Bacillus subtilis, Escherichia coli, Enterobacter aerogenes, Pseudomonas fluorescens, Serratia marcescens, Lactobacillus casei, and Proteus mirabilis were tested through a series of tests, including but not limited to:  β-galactosidase metabolism, nitrate reduction, starch/casein/starch/gel hydrolysis, catalase, oxidase, litmus/lactose metabolism, glucose fermentation, H2S/gas production, and indole formation.

II. Materials
The materials needed for this lab were as follows:

 

– platinum inoculating loop
– normal inoculating loop
– inoculating needle
– zinc dust
– test tube rack
– Gram’s iodine
– API 20E Quick Index Booklet
– 1 5 ml Pasteur pipette with pipettor
– sterile mineral oil
– disposable gloves
– Barritts A reagent
– Barritts B reagent
– permanent marker
– Bunsen burner
– 10% ferric chloride
– biohazard container
– oxidase test reagent (catalase)
– distilled water
– 5 ml sterile 0.85% saline (capped test tube)
– API 20E system

~ strip

~ incubator tray

~ cover

– 1 starch agar plate
– 1 plate count agar with milk plate
– 4 cotton swabs
– 3 tryptic soy agar (TSA) slants
– 4 nitrate broth tubes
– Kovac’s reagent
– 1 BBL Enterotube II System
– Bunsen burner lighter
– 9 phenol red broth tubes with Durham tubes
~ 3 sucrose peptone broths
~ 3 lactose peptone broths
~ 3 dextrose peptone broths
– 4 litmus milk tubes
– 3 gelatin deeps
– Nitrate test reagent A
– Nitrate test reagent B
– Becton Dickinson Enterotube II Interpretation Guide
– 1.5% hydrogen peroxide
– 1% crystal violet solution
– 20% KOH (with 3% creatine solution)
– 5% α-napthol in absolute ethanol
– 1 tryptic soy agar culture plate of unknown bacteria
– tryptic soy agar cultures of:

~ Serratia marcescens

~ Enterobacter aerogenes

~ Bacillus subtilis

~ Pseudomonas fluorescens

~ Lactobacillus casei

~ Proteus mirabilis

 

III. Methods

Oxidase Test: Part I

    The cotton swab was aseptically inserted (clam-shelling) into the tryptic soy agar plate of unknown bacteria and dragged across the surface of the culture to pick up some bacterial cells on the tip.  Two drops of oxidase test reagent (catalase) were applied to the swab, and observations were made 20 seconds later.  If the bacteria on the swab turned blue by 20 seconds after the application, the bacteria tested positive for cytochrome oxidase production.  If the bacteria did not change color, it was considered an oxidase negative test result.  If oxidase test results came out negative, this unknown bacterial culture was then placed to the side for later use in inoculation of the Enterotube II and API 20E systems.  If not, a different unknown bacterial culture had to be found and tested.

Inoculations

    Aseptic technique (relating to plates, inoculation instruments, and tubes) was strictly followed in all bacterial transfers performed in this lab, to avoid contamination.  Starch agar plates were inoculated with a single straight streak about 1” in length for each bacterium (BS, EC, and PM), using an inoculating loop.  The loop was also used for inoculating the casein agar plates; however, instead of a line, a circle-shaped spot inoculation about .5” in diameter was performed in each sector of the plate using EA, EC, and PF.

Tubes with liquid medium (including litmus milk, phenol red, and nitrate) were inoculated by swirling a loopful of the respective bacteria around in the broth.  Litmus milk tubes were inoculated with LC, EC, SM, and PF.  Nitrate broths were inoculated using SM, EC, PF, and PM.  Phenol red tubes were inoculated by categories, with each bacteria being used once for sucrose, dextrose, and lactose tubes; PM, EC, and EA were utilized for these tests.

Tryptic soy agar slants were inoculated with a single zigzag streak of an inoculating loop across the surface of the slant.  Gelatin deeps were inoculated with a single stab from an inoculating needle.

The Enterotube II was inoculated by unscrewing the caps at both ends, dragging the needle through an unknown oxidase negative bacteria culture, and using the handle to simultaneously twist and pull the needle all the way through every compartment, without completely removing the needle.  The needle was then pushed back into the tube until the tip of the needle was in the H2S/indole compartment, at which point the rest of the needle was broken off and used to poke ventilation holes in the plastic covering of the ADON, LAC, ARAB, SORB, VP, DUL-PA,UREA, and CIT compartments.

The API 20E system was inoculated according to the “First Period” procedure in Exercise 35 of the lab manual.  All inoculated media were loaded into/onto a test tube rack and incubated at 35oC for 18 – 24 hours.

———————————————— COMEBACKS ————————————————-

 

Enterotube II

    The results for the Enterotube II system were interpreted with regard to Figure 36.1 from the lab manual.  The indole test was performed by addition of 2 drops of Kovac’s reagent to the H2S compartment, using one of the Pasteur pipettes.  Results were determined after 10 seconds.  The Voges-Proskauer test was performed with the addition of two drops of 20% KOH and three drops of α-naphthol.  Results for the VP test were determined after 20 minutes.  All outcomes were recorded on the data sheet for later analysis.

API 20E

    The results for the API 20E system were interpreted according to Exercise 35 in the lab manual.  If the GLU microtube resulted in a negative glucose test, then the unknown was not a member of the Enterobacteriaceae and testing could not be continued.  A positive test (yellow, presence/absence of bubbles does not matter) meant that the following reagents had to be added in the order listed.  One drop of 10% ferric chloride was added to the TDA microtube (positive test = red-brown color, negative test = yellow color).  One drop of Barritts A and one drop of Barritts B were added to the Voges-Proskauer microtube.  Barritts B was added first, and results were observed after 10 minutes (positive = red-pink color change, negative = no color change).  One drop of Kovac’s reagent was added to the IND microtube; a positive indole result was shown by formation of a red ring within two minutes of addition of the Kovac’s.  A negative result was indicated by the formation of a yellow ring after the allotted time had passed.  The GLU tube was then examined for gas bubbles, with a positive result being the presence of bubbles and a negative result, the absence.  Two drops of nitrate test reagent A and two drops of nitrate test reagent B were added to the GLU microtube.  A positive test result showed after 2-3 minutes as the development of a red hue; a negative test involved the forming of a yellow hue after 2-3 minutes.  If the test turned out negative, a speck of zinc dust was added to the tube.  Ten minutes later the tube was observed for the development of a orange-pink color (negative nitrate reduction test result).  If the color changed to yellow instead, nitrogen reduction had occurred and nitrogen gas had been produced.  Finally, one drop of hydrogen peroxide was added to the MAN, INO, and SOR cupules.  A positive catalase test result involved the appearing of gas bubbles within two minutes.  A negative test has no such bubbles.  All outcomes were recorded on the data sheet for later analysis.

Starch Hydrolysis

    Several drops of Gram’s iodine were added to the bacterial streaks on the starch agar plate.  Any bacteria that seemed to repel the iodine, creating a clear area around the streak, were considered to have gone through starch hydrolysis (positive outcome).  Bacterial streaks that did not form a clear region around the line of growth, or beneath which the medium turned blue with application of iodine, did not hydrolyze starch (negative result).  All outcomes were recorded on the data sheet for later analysis.

Gelatin Hydrolysis

    After incubation, the tubes were tilted at a slight angle for observation of gel consistency.  If the nutrient gelatin was liquefied, gelatin hydrolysis had occurred and the bacteria in that culture was marked down as a positive result.  Solidified gelatin meant that no hydrolysis had occurred; this was noted as a negative outcome for that bacterial species.  At least the uninoculated control, if nothing else, should have tested negative.  All outcomes were recorded on the data sheet for later analysis.

Casein Hydrolysis

    Casein agar plates were examined for presence or absence of clear zones around the bacteria, called “zones of proteolysis.”  Presence of a zone indicated a positive casein hydrolysis test; the bacterial species in question was able to liberate proteases.  Absence of this clear zone meant a negative result, and the species was not able to hydrolyze casein or liberate proteases.  All outcomes were recorded on the data sheet for later analysis.

Oxidase Test: Part II

    Cotton swabs were used to pick up a small amount of each bacteria (one species per swab) growing in the TSA tubes that would later be used for catalase tests.  One to two drops of oxidase reagent were applied to each swab, and results were observed after about 20 seconds.  Like the first oxidase tests run in this lab, blue color change meant a positive result and no color change meant a negative result.  All outcomes were recorded on the data sheet for later analysis.

Catalase Slants

    Several drops of 3% hydrogen peroxide were added to each tryptic soy agar slant.  If gas bubbles appeared, the test was positive.  If no bubbles formed, results were found to be negative.  All outcomes were recorded on the data sheet for later analysis.

Litmus Milk Tubes

    After incubation, the four litmus milk tube cultures were observed for color change and curd formation at the base of the tube.  Gas production as a side effect of fermentation was also possible, but clear results (cracks or bubbles in the medium) were difficult to see.  If litmus had been fermented, the milk turned pink (positive test).  A blue-purple coloration indicated that lactose fermentation had not taken place (negative fermentation result), but protein metabolism had.  (the blue color comes from the proteins’ alkaline pH)The tube may also be white in color, from complete reduction of litmus contents.  Curd at the bottom of the tube signified the presence of casein digestion.  All outcomes seen in lab were recorded on the data sheet for later analysis.

Nitrate Broth Tubes

    Presence of gas bubbles in the nitrate broth tubes meant that the bacteria in the respective tubes had reduced the nitrate in the broth to nitrogen gas.  Absence of these bubbles could still have meant nitrate reduction had taken place, but to a nongaseous end product such as nitrite.  Five to ten drops of nitrate test reagent A and five to ten drops of nitrate test reagent B were added to each tube and mixed by finger-vortexing.  Results were almost immediately observable; a pink-red color was a positive nitrate test result and an absence of color meant a negative result.  Negative tests were confirmed by adding several grains of zinc powder or 5-10 drops of nitrate reagent C and shaking the tube.  Results from negative test confirmations were observed after 8-10 minutes.  If nitrate was present in the medium, then the broth appeared red at this point.  All outcomes were recorded on the data sheet for later analysis.

Phenol Red Tubes

    Red coloration of tubes after the incubation period could either mean no fermentation took place (no gas bubble in Durham tube) or alcohol fermentation occurred (gas bubble in Durham tube).  Any amount of yellow hue implied acid production, and any size bubble in the Durham tube meant that gas had been produced.  Respective positive and negative results were recorded on the data sheet for later analysis.

IV. Results

Phenol Red Tubes

sucrose

dextrose

lactose

gas

acid

growth

gas

acid

growth

gas

acid

growth

P.  mirabilis

+

+

+

+

+

+

E. coli

+

+

+

+

+

+

+

+

+

E. aerogenes

+

+

+

+

+

+

+

+

+

Gelatin Deeps

growth

gelatin hydrolysis

P. mirabilis

+

+

E. coli

+

B. subtilis

+

+

CONTROL

 

Catalase Slants & Oxidase Test

growth

gas

oxidase

E. aerogenes

+

+

E. coli

+

+

P. fluorescens

+

+

+

 

Enterotube II

glucose

lysine

ornithine

H2S

indole

adonitol

lactose

arabinose

sorbitol

VP

dulcitol

PA

urea

citrate

Unknown

+

+

+

+

+

+

+

+

+

+

+

+

 

 

 

 

 

Nitrate Broths

growth

nitrate reduction

S. marcescens

+

+

E. coli

+

+

P. fluorescens

+

+

P. mirabilis

+

+

 

Starch Agar Plate

growth

starch hydrolysis

B. subtilis

+

+

E. coli

+

P. mirabilis

+

Litmus Milk Tubes

growth

fermentation

curd

L. casei

+

+

+

E. coli

+

+

+

S. marcescens

+

+

P. fluorescens

+

+ (tiny spot)

 

Casein Agar Plate

growth

casein hydrolysis

E. aerogenes

+

E. coli

+

P. fluorescens

+

+

 

 

 

 

 

 

 

 

V. Discussion
This microbiology lab involved the use of rapid multitests (the Enterotube II and API 20E systems) to identify unknown bacterial specimens.  The biochemical activities of Bacillus subtilis (BS), Escherichia coli (EC), Enterobacter aerogenes (EA), Pseudomonas fluorescens (PF), Serratia marcescens (SM), Lactobacillus casei (LC), and Proteus mirabilis (PM) were studied via inoculation of various differential and/or selective cultures.

The API 20E System used in this lab was a fast, standardized, more managable test that combined conventional biochemical procedures used to identify 127 various taxa of bacteria in the Enterobacteriaceae family and other gram negative groups.  The system used microtubes to perform 22 common biochemical tests on pure cultures, which were standard to most identification procedures.  The structure of the system itself consisted of 20 chambers, each made of a microtube and depression known as a “cupule.”  Cupules helped to create anaerobic conditions – which were required for certain tests – via the addition of mineral oil, to block off the oxygen supply.  The API 20E strip is read by recording color changes after incubation; for some indicator systems, reagents were added before determining outcome.  Unknown bacteria were identified by using a chart technique similar to that on page 225 of the lab manual (questions 1 and 2) to give the unknown a seven digit profile number.  The API 20E Profile Index Booklet could then be consulted to find the bacterial name that matches up with the profile number.

Once results were completely formed for the API 20E strip and recorded, the profile was calculated to be 0774770.  The first three digits were the most crucial to the identification of the unknown, calculating seven was not entirely necessary.  The unknown culture was classified as Proteus mirabilis using the API 20E Profile Index Booklet.

The Enterotube II System was very similar to the API 20E in that it theoretically accomplished the same results.  However, inoculation of the Enterotube went much more quickly than that of the API 20E System.  The Enterotube was not as inclusive as the API 20E, though.  Only glucose-fermenting, gram negative, oxidase negative members of the Enterobacteriaceae family could be identified using the Enterotube.  The tube was divided into 12 compartments, each with a different solid agar culture medium.  Some were aerobic, and had air holes to allow the flow of oxygen to reach bacterial cells, but most were conducted in anaerobic conditions.  Enclosed in the tube was an inoculating needle specially made for the Enterotube, allowing for speedy inoculation of all 12 compartments in two swift twisting motions.  After 18-24 hours of incubation, the color changes in each compartment were recorded and interpreted according to Table 36.1 in the lab manual.  Some compartments had reagents added to them to produce the final test result, and this is what was recorded in the Results section for those particular compartments.  These results were placed into the chart from question 1 of Exercise 36 in the lab manual to obtain a five digit number that can be compared to the Enterobacter II Interpretation Guide to identify the unknown bacteria.

The Enterotube II System performed on the unknown bacterial culture in this lab was successfully carried out; results for each compartment were recorded in the Results section.  The code was calculated from the results to equal 37347, which did not match any numbers in the Enterobacter II Interpretation Manual.  Therefore, the true identity of the unknown bacteria defaulted back to the result from the API 20 E multitest system.

Phenol red broths were used to determine one of the most commonly studied biochemical characteristics of bacteria:  the ability to ferment carbohydrates.  There were three different carbohydrates used as indicators of beta-galactosidase activity in bacterial cultures.  These were known as sucrose, dextrose, and lactose; three phenol red tubes were made with each to accommodate for the three different types of bacteria used to inoculate the phenol red broths (PM, EC, and EA).  Any amount of yellow coloration (from phenol red reacting to acidic pH) in the resulting tubes after 24-48 hours of incubation indicated the presence of fermentation products.  Gas production could also be identified by the phenol red tubes, because any amount produced would be trapped inside the Durham tube.  As noted in the methods, complete red coloration of a gas-less tube denoted complete lack of fermentation, as the phenol red had not indicated any lowering of pH by acidic products. PM fermented carbohydrates in all but the lactose tube, but EC and EA fermented in all phenol red tubes.  At first glance, this could possibly have been explained by a false negative result (from an excessively large inoculum).  However, further research revealed that PM cannot ferment lactose, so the negative result was proven correct.

Gelatin hydrolysis was seen as a rather important biochemical characteristic because it could be used to assess pathogenicity of some bacteria.  Those bacteria that could hydrolyze gelatin were more likely to cause disease than those who could not, because the production of gelatinase could be correlated with the ability of a bacterium to break down tissue collagen and become more virulent.  PM and BS both tested positive for gelatin hydrolysis, while EC returned a negative test.  Of course, this result could have been an exception to the “usually more pathogenic” rule, because EC has long been known to be more pathogenic than both BS and PM.

Nitrate broths determined the ability of a bacterium to reduce nitrate.  Nitrate could be reduced into gaseous nitrogen, forming a bubble in the Durham tube, or reduction could have stopped at nitrite and other nongaseous products.  Therefore a confirmation of negative results (after addition of nitrate reagents A and B) was needed to be sure that nitrogen fixation was definitely not occurring.  This was done through the addition of nitrate reagent C and observing results after 5-10 minutes.  SM, PF, and PM all produced immediate positive (red) results for occurrence of nitrate reduction, yet EC remained clear (negative result).  A recheck was done to make sure that no false negative had occurred, and EC also tested positive after a period of eight minutes.  Therefore all nitrate broths brought back positive results for nitrate reduction, and EC, PM, PF, and SM were all confirmed to use nitrate as a terminal electron acceptor during anaerobic respiration.

The test for catalase presence in EA, EC, and PF was performed on tryptic soy agar because of its general properties as a nutrient and growth medium.  According to the results, neither EC, PF, or EA were strict anaerobes (rather they were obligate aerobes or facultative anaerobes), which corresponds with previous scientific findings.  Catalase production/activity was tested for by the addition of hydrogen peroxide to the slants, formation of bubbles meant a positive test.  Catalase catalyzed the destruction of hydrogen peroxide, which was harmful to the respiration classes that these bacterial species fell under.  When the oxidase test was performed, however, only PF produced positive results (presence of oxidase turned swabful of bacteria blue).  According to the lab manual, these oxidase enzymes in PF play a key role in the function of the electron transport system when PF cells respire aerobically:  “Cytochrome c oxidase uses oxygen as an electron acceptor during the oxidation of reduced cytochrome c to form water and oxidized cytochrome c.”

As for the casein agar plates, these differential media detected zones of proteolysis, or casein hydrolysis, around the species of bacterial colonies that were able to secrete proteolytic enzymes.  This plate was one of the most easily conducted biochemical characteristic-determining tests in this experiment, because it did not require addition of reagent or specific timing for result determination to be viable.  EA, EC, and PF all grew successfully on the agar, but only PF formed a zone of proteolysis.  Therefore PF was the only bacteria to be tested for casein hydrolysis in this lab that came back with positive results, indicating PF went through a hydrolytic reaction that produces soluble amino acids, which were then transported into the cell through proteases and catabolized to produce energy.

The starch agar plates were also a relatively simple test; all that was required was the addition of a small volume of gram’s iodine to yield immediately visible results.  Both EC and PM failed to hydrolyze starch and repel the Gram’s iodine, but BS was successful on this front.  As a result, BS was found able to rapidly hydrolyze the two constituents of starch:  amylose and amylopectin.  This hydrolysis yielded dextrins, glucose, alpha-amylase, and maltose.  The alpha-amylase is what repels the iodine and produces the clear region surrounding the starch hydrolysis-capable bacterial colony.

Litmus was a pH indicator used in this lab that was blue-purple under alkaline conditions and light pink in acidic conditions.  This pH indicator is the reason for the title of “Litmus Milk” tubes.  The milk part of the name comes from the tube’s milk-like consistency and the fact that it contains casein and lactose.  LC showed up for the first, last, and only time in the experiment in one of the litmus milk tubes.  EC, PM, and PF were used for inoculating the other three tubes.  LC, EC, and PF all produced a solid “curd” product and the base of their respective tubes, indicating casein metabolism.  However, PF did not obtain positive fermentation results, and remained blue in color.  LC, EC, and SM all went through lactose fermentation and caused litmus to change the tubes to a pink hue.  Peptonization – in which the casein would be metabolized completely into amino acids – did not occur with any of the bacteria in this experiment, but if it had, a clear brown liquid would have resulted.
VI. References
(Harley JP, 2011). Laboratory Exercises in Microbiology 8th Edition: 132-136, 147-150, 169-179, 189-192, 213-229.