These tiny organisms are called microorganisms and include bacteria, virus, fungus and protozoa. Microorganisms play a major role in many areas of everyday life. One important reason to study them is because they are often a cause of diseases. Microorganisms can live and grow in different conditions. The main goal of this lab was to identify two different bacteria from a list of 24 bacteria (Vergers manual) depending on these living conditions and their morphology. In order to do this we performed a number of biochemical tests such as differential and selective tests on the separated forms of the bacteria to obtain independent exults.
The two bacteria were called A and B. In this project, bacteria A and bacteria B were separated from the mixture using the streak plate isolation. This method consists of an agar plate providing nutrients for growth of the bacteria. After isolation, the two different cultures appear distinctively. Pure culture can then be harvested and used for the rest of the experiment to perform the different tests. Streak plate is also helpful to determine general aspect of bacteria like color of colonies, shape, size and arrangement. The pure cultures can be grown in slants containing agar which allow them to multiply and create stock.
Then, gram staining is a major step in identification of unknown. It helps scientists determining gram-positive and gram-negative bacteria and visualizing their shape. When bacteria appear purple on gram staining, they are gram- positive. Their thick pedagogical retain the crystal violet stain. When bacteria appear pink or red, they are gram-negative. Their thin pedagogical allows decolonize to wash crystal violet stain and the seafaring colors them in pinkish/ red. Microorganisms have an ability to grow and develop with or without oxygen. This ability is an important hint in identification.
Agar deep stabs are used to determine this ability. Aerobic species would grow on top of the agar whereas facultative anaerobic or anaerobic would grow deep in the agar. Selective media ensure growth of specific species of bacteria and inhibit growth of the others. This is why uses of selective media is important to identify unknowns. Selectivity depends on components of the media and living conditions of microorganisms. Four different selective media were used, Penitently Alcohol Agar (PEA), Desolately Agar (AD), Imitation Salts Agar (MS), Close Lysine Desolately Agar (SLD), and Eosin Methyl Blue Agar (EMBED).
PEA media is selective for gram-positive Cisco as streptococcus and staphylococcus. These organisms are not inhibited by penitently alcohol. It inhibits gram negative bacteria. MS is composed of Sodium Chloride in high concentration. It then selects bacteria that do not undergo dehydration. This media kills most of listed bacteria except staphylococcus. Plus, color of the plate after inoculation informs about which species it is. A yellow growth represents an organism that can produce acid whereas a red growth isn’t.
AD is composed of desolately citrate which inhibits the growth of gram positive bacteria. A change of color informs about PH. A red color signifies a pH of 6. 8 and below and a yellow color signifies a pH of 8. 0 and up. SLD agar selects for shillelagh and salmonella species. The agar contains sodium desolately that inhibits the growth of other bacteria. Color of the growth helps identify further, a pink growth is a shillelagh species whereas a pink/ red growth is a salmonella species producing ferric ammonium citrate precipitate and reduction sulfur to hydrogen sulfide.
Red growth without black precipitate means the bacteria do not ferment the close and alkaline products from hygiene decertification. EMBED agar is selecting either gram-positive or negative organisms. Agar color after inoculation means the bacteria is not fermenting lactose and use sucrose. Black color of the plate after inoculation means the bacteria ferments lactose. Moreover, differential tests are used to differentiate microorganisms. They differentiate bacteria in function of their biochemical properties. MRS.. Tests are called Methyl Red (MR..) and Vogues-Prorogues (UP).
MR.. Test is used to determine ability of bacteria to oxidize glucose and color change from yellow to red determine their ability of fermenting acids. UP test detects organisms that ferment glucose and convert acids into action and 2,3-butadiene by a red color change. Sulfide Indolent Motility or SIMI test is used to determine if a bacteria is motile by observing growth within the media, their ability to reduce thyrotrophic inducing producing indolent and pyrrhic acid ammonia in observing red layer on top and finally their ability to reduce sulfur to hydrogen sulfide by observing a black color change.
SIMI is differential for enteric organisms, it is used for intercontinental. Triple Sugar Iron agar or ITS is used to differentiate acetate on their ability to reduce glucose, lactose, sucrose, to reduce sulfur and to produce gas. Glucose fermentation is seen by a color change, the slant would be yellow with red color on surface. The bacteria is fermenting glucose, sucrose and lactose if the slant is all yellow. If black color is present, the bacteria is producing sulfur. Gas production is represented by cracks in the agar.
Citrate test is testing the bacteria for its ability to utilize citrate as a carbon source. The slant stays green at neutral pH but is turns blue if it uses this citrate and the pH raises above 7. 6. It differentiates intercontinental family. Catalane test indicates if a bacteria is producing catalane and cellular toxins by adding drops of hydrogen peroxide on bacteria. If it produces bubbles when adding the drops, test is positive and bacteria produces catalane. Oxides test determines the ability of a microorganism to produce stockroom c oxides.
It is determinate by a blue coloration 15 seconds after adding oxides reagent on bacteria. Finally, gelatins test determines the ability of bacteria to utilize gelatin as a carbon source. If the bacteria uses gelatin as a carbon source, the gelatin results in a quid state. If it does not use gelatin, the gelatin stays solid. It is a hydrolysis. This project aims to identify unknown bacteria A and B with the help of the tests previously presented and by comparing them to the list of known species. To do this comparisons of the results and identify the unknowns, the Vergers manual will be used.
MATERIALS AND METHODS The materials used when performing the experiments were: – Bunsen burner – Gloves + lab coat – Ethanol, Del water – Inoculation loops/needle – Nutrient agar plate – Tropic soy agar slant – Close-Lysine-Desolately agar – Eosin-Methyl-Blue agar Sulfide Indolent Motility medium – Imitation salt agar – Triple sugar slant agar – PEA agar plate – Desolately agar – Citrate agar slant – Gelatin – Grams stain kit (crystal violet, seafaring, iodine, and decolonize) – Oil immersion – Microscope – Slides – Tray (for test tube) – Indolent reagent (Kava’s reagent) – UP reagents – Catalane reagent – Oxides reagent – q-tips The unknown number 68 handed out by the teacher on February 18th contained both a gram-positive bacteria and a gram-negative bacteria. The first step to differentiate the unknowns was to separate the two bacteria. In order to separate the two bacteria from the mixed culture, the streak plate method of isolation was used. This method reveals single species known as pure culture and colonies of single species.
An inoculating loop was sterilized with a Bunsen burner and put into the unknown mixture. After removal with bacteria on the loop, the quadrant streak method was performed on the agar plate (Lowlife, Pierce). The streak plate was then incubated face down at 37 degrees Celsius for 48 hours. After observing the plate, another plate was streaked to further isolate the colonies and give better results. The plates were labeled with the date, medium, name of student and section number. A portion of each isolated colony had then been transferred into sterile tropic soy agar slant to start a pure culture. The two slants were labeled with the date, medium, name of the student and section number.
The tubes were incubated at 37 degrees Celsius for 48 hours and then placed in a rack at room temperature for the rest of the lab. The plates and slants were furnished with enough nutrients and water to provide good growth of the bacteria for several weeks out of the incubator. The second step in identifying a acetate is to perform a gram staining. It helped detecting differences in shape and size between organisms. The gram stain procedure was performed as directed in the lab manual (Lowlife, Pierce). Imitation salt agar, desolately agar, phenyl alcohol agar and nutrient agar were inoculated on March 18th following the same process. The four plates were divided in half and labeled. One half was bacterium A and the other half was bacterium B.
The pure cultures from the agar slants were used to inoculate the four plates. The plates were labeled on the bottom with medium, name of the student, date, class number, section number and unknown number. The following process was repeated for each plate: a loop was sterilized using the Bunsen burner. The loop was then used to transfer bacteria A from the agar slant to one side of the plate. The plate was inoculated with a gig gag motion of the loop down the half of the plate. The loop was then sterilized in the flame. This procedure was then repeated for bacteria B on the other half of the plate. All plates were incubated face down at 37 degrees Celsius for 48 hours.
SLD agar and EMBED agar were inoculated and incubated on March 25th following the same process as previously enunciated. SIMI agar tubes were inoculated on March 25th. One tube contained bacteria A and the other tube bacteria B. The test tubes were labeled with bacterial content and inoculated from the agar slants pure isolated colonies. A needle was sterilized using the open flame and used to transfer each bacteria from the slants to the test tubes. The agar was stabbed deeply with the needle and the needle sterilized again. The SIMI test tubes were incubated at 37 degrees Celsius for 48 hours. After incubation, five drops of Kava’s reagent were put in each test tube using a sterile pipette. MRS.. Tests were performed on March 25th.
To do so, a sterilized loop was used to transfer the bacteria from the slants to the tube containing nutrient broth. There was two tubes, one for bacteria A and one for bacteria B. The tubes were labeled with the type of bacteria, date, name of student and relevant information. The tubes were placed in the incubator at 37 degrees Celsius for a week. After incubation, the content of each tube was separated in half in two other small test tubes in order to have two tubes containing bacteria A broth and two tubes containing bacteria B broth. The tubes ere labeled with the contained bacteria and tests to be performed (either MR.. Or UP). For the MR.. Test, five drops of methyl red reagent were added to the tubes and a color change was noticed or not and recorded.
For the UP test, 15 drops of reagent A and five drops of reagent B were added to the tubes and the tubes were shaken regularly for one hour. After one hour a color change or not was recorded. ITS agar slants were streaked similarly as tropic soy agar slants so following the procedure in the lab manual (Lowlife, Pierce). However, one step was added, the agar was also deeply stabbed with a sterilized needle. The needle was removed and sterilized and the tubes were labeled and incubated at 37 degrees Celsius for 48 hours. Citrate slants were inoculated following the same process as agar slants then following the procedure in the lab manual (Lowlife, Pierce). The slants were labeled regarding each bacteria and incubated at 37 degrees Celsius for 48 hours.
Gelatin hydrolysis test was performing using a sterilized needle. The sterilized needle was used to transfer bacteria from pure culture slants to gelatin tubes. The needle stabbed gently the gelatin without the tip of the needle touching the bottom of the tube. The tube was labeled and the process repeated for the second bacteria. The gelatin tubes were kept in a rack at room temperature for one week. For the catalane test, a sterilized loop was used to transfer the bacteria from the agar slants to a cleaned and sterilized slide. The bacteria was applied on the slide making a smear. Few drops of hydrogen peroxide were added on the smear. Presence or absence of bubbles was recorded.
The process was then repeated for the second bacteria For the oxides test, a sterilized loop was used to transfer a sufficient amount of bacteria from the agar slants to the IP of a q-tip. Using a sterile pipette, few drops of TEMP were added on the tip of the q-tip. Any color change was recorded after 15 seconds. This process was repeated for the other bacteria. RESULTS Bacteria A Bacteria B PEA Growth Huge growth MS No growth Growth, no color change False positive Pink/red color SLD Pink/red Black precipitate EMBED Black SIMI Yellow MR.. Indolent production Catalane ITS Red Citrate Blue Green Gelatin Oxides Table 1: Table of the results of all tests performed Figure 1: showing gram positive rod bacteria B under microscope, oil immersion 1 ox
Figure 2: showing gram negative rod bacteria A under microscope, oil immersion Figure 3: showing result on nutrient agar of bacteria A (left) and B (right) Nutrient agar is used to visualize general aspect of bacteria. Figure 4: showing result on penitently alcohol sugar of bacteria A (left) and B (right) Huge growth of bacteria B on PEA Figure 5: showing result on desolately agar of bacteria A (left) and B (right) Color change means bacteria A is a lactose ferment. Figure 6: showing result on imitation salt agar of bacteria A (left) and B (right) Bacteria B should not grow on MS, then this is a false positive result. Figure 7: showing result on eosin methyl blue agar of bacteria A (left) and B (right) Color change means bacteria A is a lactose ferment.
Figure 8: showing result on SLD agar of bacteria A (left) and B (right) There had been contamination of all plate by one of the organisms. Figure 9: showing result of SIMI on bacteria A Bacteria A is producing sulfur. Figure 10: showing result of SIMI on bacteria B Bacteria B does not produce sulfur. Figure 11: showing negative result of UP on bacteria A Figure 12: showing negative result of UP on bacteria B Figure 13: showing positive result of MR.. On bacteria A Figure 14: showing negative result of MR.. On bacteria B Figure 15: showing result of ITS on bacteria B Bacteria B is fermenting glucose. Figure 16: showing result of ITS on bacteria A Bacteria A is fermenting glucose, sucrose and lactose.
Flowchart Gram stain Gram negative rod Oxides (positive) Lactose fermentation (positive) Indolent (negative) Motility (positive) HAS (positive) Salmonella typographic Figure 17: Flowchart showing deduction for unknown bacteria A Figure 18: flowchart showing deduction for unknown bacteria B DISCUSSION The plates streaked at the first step with the mixed culture and incubated beveled two distinct types of colonies. There was a small round yellowish colony and the other colony was very cloudy, white and larger. The pure cultures of the bacteria remained sealed with Paraffin during the whole time of the project to exclude other organisms to contaminate and grow on the nutrient slant. Plus, all the materials used were sterilized with heat or ethanol before and after using it to avoid contamination of the slants and tests by microbes possibly present in the air.
The bacteria B that came from the small round yellowish colony was tested with gram stain. The result (see figure 1) showed purple rod which significance that it was a gram positive bacteria. Gram positive bacteria have a thick pedagogical layer. That is why they appear purple when observed under the microscope. They are able to retain crystal violet and not let the decolonize wash it. The bacteria A that came from the cloudy colony was also tested with gram stain. The result showed on figure 2 revealed pink rod, that significance that it was a gram negative bacteria. Gram negative bacteria have a thin pedagogical layer, which is why they appear pink.
The decolonize washed the crystal violet cause the pedagogical is unable to retain in. Then the countersink seafaring colored it in pink. As seen in figure 4, both bacteria grew on PEA which was not possible as it was selective for only gram positive Cisco so this result was not relevant to determine the unknowns. The bacteria was not streptococcus or staphylococcus. The desolately agar revealed that bacteria A was able to ferment lactose. It was noticed by the action of neutral red, a pH indicator which changes the color from yellow to pinkish at a pH of 6. 8 (See figure 5). Figure 6 showed that bacteria B grew on imitation salt agar but without a color change.
This result was considered as a false positive so the unknown was not a staphylococcus. Figure 7 showed bacteria A growing black on eosin methyl blue which meant that bacteria A was a lactose ferment. This test was aimed to reveal bacteria such as salmonella and shillelagh species. Bacteria A was turning black on SIMI deep agar stab (see figure 9) which meant that it was a motile sulfur reducing bacteria. Bacteria A had a positive result to catalane test. Bacteria A was then able to produce cellular toxins. It also had positive results for MR.. (figure 13) and oxides tests. Positive result for oxides implied that bacteria were able to produce stockroom C oxides.
When grown on ITS (figure 16), the medium stayed yellow meaning that the bacteria was able to ferment glucose, sucrose and lactose. Figure 8 showed the result on SLD agar which was all yellow. This plate was told to be contaminated and so streaked again. The new plate showed bacteria A growing pinkish with a black precipitate on non-contaminated SLD agar. This result was leading to conclude that bacteria A was salmonella typewriting as SLD was a selective and differential medium for almoner species which produced ferric ammonium citrate. Bacteria B was a gram positive rod as seen in figure 1. It is already known that bacteria B grew on MS (figure 6) without color change but this result was recorded as false positive.
Plus, bacteria B didn’t grow on desolately agar (figure 5) or EMBED agar (figure 7) so this microorganism wasn’t fermenting lactose or sucrose. Bacteria B wasn’t motile (figure 10) as there was no color change in the SIMI test. MR.., UP, indolent, catalane and citrate tests were all negative. However, the bacteria grew on ITS Lana and showed red color on the surface and yellow color deeper (figure 15) which suggested that the organism ferments only glucose. Plus, oxides test for organism B was positive, then it was able to produce stockroom C oxides. The results from these tests suggest that bacteria B was a bacillus species. However, further tests would have needed to be done to determine which specific species of bacillus was present in the mixed culture.
Testing presence or absence or spores and leading acid-fast reactions could have been helpful to differentiate one bacillus species from another one. Other selective tests could eave been conducted because these tests only promote the growth of one or two organisms so each species would have been differentiated. All plates and slants were incubated for 48 hours. Indeed, a longer incubation time would have been deleterious for the efficiency of the test. Bacteria have an exponential growth rate, since the plate was divided in half, a longer incubation time could have led one bacteria to overgrow and take over the plate and then leading scientists to a wrong result. Other tests like gelatins required a longer incubation period.