Here is an example of a fairly good lab report handed in around 1995. This person augmented her lab report with photos, but that is certainly not a requirement for a lab report in this course. One criticism of this report would concern the lack of references for the hard facts in the text of the report!
Enrichment and Isolation of
Purple Non-Sulfur Photosynthetic Bacteria
Bacteriology Lab 102
April xx, 199x
Phototrophic (or photosynthetic) bacteria have been studied for over 100 years. These organisms play an important role in the anaerobic cycling of organic matter – as producers (photoautotrophs, utilizing CO2 as their carbon source and H2 as their electron donor), and as consumers (photoheterotrophs, using organic molecules as their carbon source and electron donor).
Photosynthetic bacteria can be classified into two distinct groups:
- anoxygenic phototrophic bacteria
- oxygenic cyanobacteria
In the first classification, there is further subdivision into the green sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria, all of which have a unifying characteristic in their ability to grow phototrophically (i.e., utilizing sunlight as a mode of energy generation) under anaerobic conditions. The distinguishing characteristic of the purple non-sulfur bacteria that we will study in this experiment, is that they do not demand a high concentration of sulfide in order to thrive – hence the name non-sulfur bacteria – even though all organisms need some sulfur for growth.
The presence of phototrophic bacteria can be detected in natural environments by the appearance of pigmented "blooms," or colored waters, indicating the growth of photosynthetic organisms. Purple non-sulfur bacteria exist mainly in anaerobic conditions that receive light, such as at the bottom of water sources. However, evidence of purple non-sulfur bacteria is difficult to detect because they exist naturally in low numbers, rarely creating dense blooms. In natural environments, their coexistence and competition with heterotrophic microbes make it difficult to obtain organic substrates used as carbon and energy sources, thus their growth is inhibited. (The low concentrations of purple non-sulfur bacteria are also most likely due to their sensitivity to sulfide concentrations in the water – see #5 below).
In the absence of blooms, however, water can be collected and samples incubated under conditions suitable for the enrichment of certain strains of photosynthetic bacteria. It is the purpose of this experiment to cultivate and isolate purple non-sulfur bacteria by selectively enriching the media used to provide good growth conditions and create additional selective pressure from various parameters, in order to narrow the range of strains seen. (Representative strains of purple non-sulfur bacteria observed in this experiment include morphological varieties of rods, cocci, and spirilla.)
The use of a combination of conditions needed to cultivate purple non-sulfur bacteria that are utilized in this experiment include the following:
- Oxygen requirement: Purple non-sulfur bacteria are a group of gram negative, facultative anaerobes. (Oops, remember that oxygen relationships as we have defined them – based on Bergey's Manual definitions – apply only to chemotrophic organisms. These organisms are best called "facultative phototrophs" as discussed here.) They are, however, tolerant to an oxygen atmosphere to some degree and can also grow as respiring chemoheterotrophs in aerobic conditions. (In this situation, photosynthesis is inhibited by the presence of oxygen and the production of pigment is suppressed) By anaerobic incubation in this experiment, we inhibit any chemoheterotrophic growth. (Better to say: The medium utilized in this experiment contains nothing that promotes growth under anaerobic conditions due to fermentation or anaerobic respiration. Anaerobic incubation also does not allow for aerobic respiration. So, growth of organisms that perform anoxygenic phototrophy is promoted and they are detectable by their photosynthetic pigments.)
- Temperature: Isolation of purple non-sulfur bacteria require temperatures of 30 degrees centigrade, so monitoring of growth temperature is important. (This needs a reference. Sometimes over a spring break we may incubate the plates at room temperature.)
- Light source: The light requirement for the growth of purple non-sulfur bacteria is of significant importance as it relates to the optimal temperature conditions. As a standard light bulb will generate heat and increase the temperature conditions of incubation, it is essential to utilize a tungsten light source which does not produce additional heat. (The main thing about a tungsten light concerns the optimal wavelengths of light that the desired organisms find relatively more abundant than in sunlight.)
- Sulfide concentrations: Conditions which favor a low concentration of sulfide will support the growth of purple non-sulfur bacteria as they do not demand high concentrations of sulfide. (bacteria that do demand high concentrations of sulfide – photoautotrophs – namely the purple and green bacteria are thus inhibited).
In addition to the above, the use of a mineral salts solution (which is known to support the growth of photosynthetic organisms) was employed. To this solution, ammonium chloride was added (as a Nitrogen source), yeast extract (supplies B vitamins required by some species for growth), and sodium succinate (an organic compound which is not used as a carbon/energy source by most other chemotrophic organisms).
MATERIALS AND METHODS
Procedural methods follow the outline of Experiment 11.1 in the General Bacteriology Lab Manual, pages 55-56. Modifications to the experiment include the addition of two extra water samples from various locations, so that results can be compared. Water samples were collected from the following locations:
- Central Park – New York City, New York (Collected 3/11/96)
- Yahara River – Madison, Wisconsin (Collected 3/ ? /96)
- Blue Ridge Mountains – Blowing Rock, North Carolina (Collected 3/14/96)
Additionally, after growth was obtained on succinate agar plates, selected colonies from each sample were inoculated onto two slants of succinate agar and incubated in both aerobic conditions (30 degrees) without light, and anaerobic conditions (30 degrees) with light.
Initial results from the first step of the experiment (after anaerobic incubation in 30 degrees for 4-7 days under a tungsten light source) yielded three bottles of photosynthetic growth in varying degrees. The bottle containing the water sample from Central Park appeared to have manifested the most growth as it displayed an overall dark purplish color. The bottle containing the water sample from the Yahara River also displayed an overall color, but it was of lighter value with an orange tone. The third bottle containing the water sample from the Blue Ridge Mountains was clear with just a small amount of purplish color/growth at the bottom.
Diagrams of the three enrichment bottles were inserted here, showing degrees of turbidity.
Wet mounts from the bottles of bacterial growth displayed a variety of tiny microorganisms – it was difficult to identify specific organisms as there was an abundance of movement (mostly brownian, but there seemed to be some organisms that were actually swimming in the solution). The majority of bacteria looked to be of an oval or round nature in shape, particularly those found on the slides from the Yahara River and Blue Ridge Mountain samples. The Central Park wet mount displayed organisms that appeared to be strung together or looked like curved rods.
Streaking of succinate agar plates with all three samples produced the following results:
Photographs of plates from the three enrichments were inserted here. (Again, photos are not required.)
After inoculation and incubation of the succinate agar slants, it was noted that the slants which were incubated in anaerobic conditions (30 degrees with light) produced the most growth from all three samples. (Adequate growth was noted for the Central Park and Yahara River samples, while the Blue Ridge Mountain sample showed only a small amount of growth). The slants from all three samples incubated in the aerobic conditions (30 degrees without light) produced growth that was barely detectable and in some cases did not produce any growth at all (Blue Ridge Mountain sample). Observations of the plated cultures, colonies, and bacterial strains found in each sample are tabulated as follows:
||incubation w/succinate broth
||colonial characteristics after plating on succinate agar
||microorganisms seen in wet mount
||Lots of particulate matter
||Opaque, dark purplish
||Very small, round dark purplish colonies with a few white ones – SOFT
||Mostly elongated forms with budding ends
||Lots of particulate matter
||Very small, round orange colonies w/ a few white ones – SOFT
||Oblong bacteria w/segmented middle – binary fission
|Blue Ridge Mountains
||Clear w/small amount purple growth
||Small, round off-white colonies w/ some transparent ones – SOFT
||Elongated w/buds and oblong, segmented bacteria
Apparently only one colony type was studied from each of the three samples. For more than one isolate from any sample, we would require the observations of colonial characteristics and wet mount on a separate row for each isolate.
(Note: Observed strains were possibly identified as Rhodopseudomonas for the Central Park sample, Rhodobacter for the Yahara River sample, and the Blue Ridge Mountain sample appeared to contain morphologically similar species of both Rhodopseudomonas and Rhodobacter. A pure colony would have only one type. It is important to point out that although those were the only distinguished organisms in the slide preparations, there most likely were numerous types of bacteria in each sample.)
Successful cultivation and isolation of purple non-sulfur bacteria in this experiment appears to have been accomplished, as evidenced by the large blooms and the observation of species strains in wet mounts preparations. This would seem to indicate that the water samples studied did contain purple non-sulfur bacteria although they were not exposed until suitable growth conditions were provided (i.e. the inhibition of photoautotroph and chemoheterotroph competition by the incubation of samples in anaerobic environments and the use of sodium succinate as an organic compound source for carbon and energy) and they were able to flourish.
The variety of sample locations poses the question as to whether purple non-sulfur bacteria have any habitat restrictions (e.g. altitude, pollution, etc.) or are able to grow anywhere there is an adequate supply of growth factors. There certainly was production of the bacteria in the Central Park and Yahara River samples as evidenced by the colorful blooms and culture growth. The Blue Ridge Mountain sample, however, which was collected from approximately 4000 feet altitude, showed the least amount of bloom and had non-pigmented colonies (even with selective enrichment) – which leads me to wonder whether purple non-sulfur bacteria may not be able to flourish at high altitudes, or is able to grow but possibly in another form – maybe the culture is not purple non-sulfur bacteria at all (?). (With only a small amount of experience in this field I cannot draw any conclusions)
Also, it should be noted that the experiment had observable results because of the fact that our initial water samples were first placed in an enrichment broth (sodium succinate/mineral salts solution) and incubated anaerobically which allowed any purple non-sulfur bacteria to flourish without the competition of other organisms that would normally be present in an aerobic or anaerobic environment in nature or one with a higher sulfide concentration.
Anaerobic Microbiology – A Practical Approach. P.N. Levett, University of West Indies, I.R.L. Press, New York, New York, pp.247-273, 1991.
Biology of Anaerobic Microorganisms. Zehnder, Alexander J.B., Agriculture University, The Netherlands, John Wiley and Sons, New York, New York., pp.39-79. 1988.
General Bacteriology – A Laboratory Manual. Lindquist, John, University of Wisconsin, Madison, McGraw-Hill, Inc., pp.55-56, 1994.