Spore formation
Plate cultures were generated from three isogenic strains of B. subtilis and patched onto one agar plate in short streaks to test their ability to form normal germinating spores. Sporulation in B. subtilis has been used as an excellent model system to study cell differentiation for almost half a century. This research has given us a detailed picture of the genetic, physiological and biochemical mechanisms that allow bacteria to survive harsh environmental conditions by forming highly robust spores9. Spore formation in B. subtilis has been classically viewed as an example of unicellular differentiation that occurs in response to nutritional starvation10. Hence after a week’s incubation at 37°C the bacteria cultured had exhausted their nutrients and begun to differentiate to form spores. To observe this, the spores must be induced to germinate as they would have in nature and this is done in the laboratory by heat-activation allowing for synchronous germination of spores11. An 8cm paper disc was allowed to adhere to the colonies for a minute and marked to correspond to the relevant strains on the agar plate. It was then transferred to a Heat shock plate which is used for germination plate tests and incubated at 65°C for 2 hours. It was then transferred to Tetrazolium agar, also used for germination plate tests, and incubated at 37°C for 60 minutes12. This enabled the colonies on the disc to change to a colour corresponding to their ability or inability to form spores.
Two strains of B. subtilis, PY79 and SC1988 grown overnight were used to quantify heat resistant spores by serially diluting each. 1 ml of each and transferring to a microfuge tube which was incubated in an oven at 65°C for 45 minutes. During this time the 2 unheated cultures were serially diluted to 10ˉ4 to 10ˉ8 on nutrient agar plates and dilution was carried out with sterile water13. Serial dilutions are carried out to avoid too many colonies from developing on the plate14 and need to be done several times in order to achieve a point where the viable cells are possible to quantify.
Chemotherapeutic agents are substances that are selectively toxic and only act on selected substances. Gram positive bacteria, such as B. subtilis tend to be more sensitive to chemotherapeutic agents as compared to Gram negative bacteria, such as E. coli., perhaps due to the outer membrane present in Gram negative bacteria which provides extra protection. Chemotherapeutic agents come in many forms, including antibiotics which were used in this practical. Antibiotics are substances produced by microbes and kill or harm other microbes. They are used to treat infections caused by bacteria by killing or preventing bacterial growth without harming the host. These antibiotics include the aminoglycosides, macrolids and the tetracyclines, some of which were used in this experiment. Erythromycin for example, is a macrolide antibiotic and contains large lactose rings connected to sugar molecules. The macrolides account for 11% of the total world production and use of antibiotics15.
Other antibiotics used here include penicillin or penicillin G, the first antibiotic discovered and also the first clinically effective. It is dramatically effective in controlling staphylococcal and pneumococcal infections and also more effective for the treatment of streptococcal infections as compared to sulfa drugs. Penicillin G is active primarily against Gram positive bacteria as Gram-negative bacteria are impermeable to the antibiotic16. Ampicillin, another antibiotic used, is mainly effective against Gram-negative bacteria and is different only from penicillin due to the presence of an amino group17.
An agar diffusion test was used to observe the effectiveness of a several antibiotics to a B. subtilis by impregnating filter paper discs with the antibiotics to be tested and putting them on the agar where the bacteria was being cultured. The effectiveness of each antibiotic against a particular bacteria is determined by observing the radius of inhibition zone around each antibiotic disc as this indicates the susceptibility of the bacteria in relation to that antibiotic, i.e. the larger the radius, the more effective that antibiotic is against that particular bacteria.
The human body has hundreds of species and billions of individual microorganisms growing on or in it, known as the normal microbial flora18. It is therefore of great interest to be able to examine some of these bacteria and even attempt to identify them. This was done by taking samples of swabs from the throat and nose using sterile cotton swabs and culturing them on blood agar and Vogel-Johnson respectively and incubated at 37°C for a week. Then both cultures were examined and their appearance recorded [Fig. 1.7]. Both also underwent the Gram stain [Fig. 1.8 & appendix III]. They were then re-streaked on nutrient agar, blood agar and Baird-Parker agar and incubated at 37°C19. Re-streaking is usually performed in order to obtain a pure culture provided the colony is well isolated. The re-streaked cultures underwent the same procedures as the original cultures and their appearances and structures recorded [Fig. 1.9]. However, in order for specific identification, the bacteria also underwent some diagnostic tests:
The catalase test is used to identify organisms which produce the catalase enzyme, an enzyme which attacks hydrogen peroxide by converting it to water and oxygen gas, thereby helping to protect bacteria against hydrogen peroxide which is harmful to the cell’s components20:
H2O2 + H2O2 → 2 H2O + O2
This is also a useful test for distinguishing Staphylococci which are catalase-positive(+ve) from Streptococci which are catalase-negative(-ve)21. When a catalase-+ve organism is exposed to hydrogen peroxide, the hydrogen peroxide will bubble. The nose and throat cultures from the nutrient agar plate went through this test.
The oxidase test is done to test for aerobic microorganisms by checking for the presence of the electron transport chain which is the final stage of aerobic respiration with oxygen being the final electron acceptor in the chain. The oxidase reagent contains a chromogenic reducing agent, which is a compound that changes colour when it becomes oxidized. If the microorganism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds21. Nose and throat cultures from the blood agar were used to undergo the oxidase test.
Slide agglutination tests were conducted for Staphylococcus and Streptococcus suspects, with a separate test for each. A positive result (i.e. agglutination occurs) in the strep-test confirms the bacteria as Streptococcus while a positive result in the staph-test confirms the bacteria as Staphylococcus aureus (see results).
Results
Results
Streaking on plates:
Below are results of the streaking exercise in which two strains of B. subtilis were streaked onto agar plates in order to generate single colonies of bacteria:
Figure 1.1: test for single colonies in PY79 and SC1988 strains of B. subtilis:
As can be seen, the PY79 strain produced single colonies whereas the SC1988 did not.
Spore formation/germination:
[See appendix I for diagram of coloured paper disc]
This part required testing for spore germination using a paper disc to produce various colours depending on whether the strain of B. subtilis was able to form spores or not.
It was found that the PY79 strain turned a clear pink colour indicating that it was able to successfully form normal spores (Ger+). Strain SC2376 turned a pink-yellow colour indicating partial spore formation but suggesting the inability of the spores to germinate fully (Ger±). Strain SC1988 however remained colourless/white indicating that it was unable to form any proper spores (Ger-).
Quantification of heat resistant spores:
Here, two strains of B. subtilis, PY79 and SC1988 grown overnight were used to quantify heat resistant spores by serially diluting in sterile water from 10ˉ4 to 10ˉ8 on nutrient agar. ~1 ml of each strain and transferred to a microfuge tube and incubated in an oven at 65°C for 45 minutes. During this time the 2 unheated cultures were serially diluted to 10ˉ4 to 10ˉ8 on nutrient agar plates. After incubation the heat-treated cultures were also diluted with 10ˉ4 - 10ˉ8 being plated for strain PY79 and10ˉ0 - 10ˉ2 for SC1988.
The table on the next page displays the viable count for all the diluted cultures.
Results for PY79
Figure 1.2: table displaying colonies and c.f.u. of heat and unheated cultured of PY79 strain:
As can be seen from the above table, some of the colonies could not be counted due to their extensively large size hence the c.f.u. value for them could also not be determined.
Results for SC1988
Figure 1.3: table displaying colonies and c.f.u. of heat and unheated cultured of SC1988 strain:
In the case of SC1988, there seems to have been a problem with the aseptic technique when culturing the bacteria due to which contamination occurred hence some of the cultures not having grown at all.
Comparison of microbial sensitivity to various chemotherapeutic agents:
In this part the effectiveness of several different antibiotics was tested on culture of B. subtilis in an agar disc diffusion test. Several paper discs were impregnated with the antibiotics to be tested and put on the agar. The effectiveness of each antibiotic against a particular bacterium is determined by observing the radius of the inhibition zone around each antibiotic disc as this indicates the susceptibility of the bacteria in relation to that antibiotic, i.e. the larger the radius, the more effective that antibiotic is against that particular bacteria. Below are the measurements of the inhibitory zones of all the antibiotics used:
Figure 1.4: table displaying radius (mm) of inhibition zone in presence of Ampicillin:
Figure 1.5: table displaying radius (mm) of inhibition zone in presence of Erythromycin
Figure 1.6: table displaying radius (mm) of inhibition zone in presence of different antibiotics
Analysis of human flora:
Samples of human flora were taken from the nose and throat and cultured on blood agar and Vogel-Johnson plates respectively. These were incubated at 37°C. The appearance of both cultures is recorded in the table on the next page as well as the appearance of the re-streaked cultures on the three different types of agar.
Figure 1.7: table displaying information observed about nose and throat culture:
Diagnostics: Gram stain
After observation of the colonies, they were Gram stained to determine their structure, i.e. whether they were Gram positive or Gram negative [see appendix III]. The bacteria were observed by phase microscopy to identify certain morphological details such as their arrangement and shape. Below is a table displaying the appearance of bacteria from both cultures:
Figure 1.8: table displaying observations of nose and throat culture bacteria after Gram staining:
As can be seen from above, both the nose and throat cultures tested positive in the Gram-stain.
Colony re-streaking:
The nose and throat cultures were then restreaked onto three different agar plates: blood agar, Baird-Parker and nutrient agar. Below is a table displaying the appearance of each restreaked culture as observed by the naked eye:
Figure 1.9: table displaying information observed about re-streaked cultures of nose and throat:
Diagnostics: Catalase Test
In addition to Gram staining and re-streaking the bacteria, they were also put through some simple biochemical tests such as the Catalase test which is explained in greater detail on page 4. The bacterial colonies were taken with a sterile loop and smeared onto a microscopic slide to which a drop of hydrogen peroxide solution was added. If bubbles are produced, the reaction is Catalase positive:
Figure 2.1: table displaying observations of nose and throat culture bacteria in the Catalase test:
Diagnostics: Oxidase Test
The catalase test was followed by the oxidase test which tests for the presence of aerobic organisms. More information on this test can be found on page 5. Below are the results obtained from the oxidase test conducted on both nose and throat cultures:
Figure 2.2: table displaying observations of nose and throat culture bacteria in the Oxidase test:
As can be seen from the above tables, both the nose and throat cultures tested positive for the catalase test but negative in the Oxidase test by not changing colour.
The nose culture tested negative in the staphyltect. test thus it was clear that the bacteria were not Staphylococcus aureus. The bacteria shares much of its morphology with Staphylococcus epidermidis
The throat culture was put through the staphyltect. agglutination test to determine its genus and tested positive thus confirming that it is most likely a Staphylococcus aureus bacteria.
Discussion
Staphylococci are perfectly spherical cells about 1µm in diameter. They grow in clusters due to dividing in two planes. This configuration helps to distinguish staphylococci from streptococci, which are slightly oblong cells that usually grow in chains (because they divide in one plane only). The Catalase test is important in distinguishing streptococci (catalase-negative) from staphylococci, which are vigorous catalase-producers23.
Both the nose and throat bacteria tested positive in the Gram stain indicating that both are Gram positive bacteria. As the colonies of both the cultures were circular, the nose and throat samples cultured on blood agar underwent the catalase-test and after testing positively, underwent the oxidase test to which they both tested negative. The agglutination tests have shown both bacteria to be of the genus Staphylococci, a Gram-positive spherical bacteria. These occur in microscopic clusters resembling grapes and only Staphylococcus epidermidis and Staphylococcus aureus are significant in their interactions with humans.
S. aureus can cause infections such as boils and styles, i.e. ‘pus-forming’ infections as well as food poisoning by releasing enterotoxins into food. Hospital strains of this bacteria are resistant to a variety of antibiotics with the infamous MRSA bug being a strain of S. aureus24. Phagocytosis is the main mechanism of combating staphylococcal infections at the moment.
S. epidermidis is non-motile and a facultative anaerobe. It is mainly found on the skin and mucous membrane of warm-blooded animals and can be a pathogen25
