EXPERIMENT 1- SUBDIVISION OF MICROORGANISMS IN GROUPS

Introduction and aim Investigation of the microbiological quality of food products is usually done by culturing microorganisms from food. Pure culture of microorganisms is required for the further examination of isolated microorganisms, which is done by streak plate technique The aim of the experiment is to obtain pure cultures from provided strains and to examine the pure cultures further (microscopy, biochemical reactions) in order to divide the strains in groups.

Materials and methods Sterile inoculating loops Six strains(agar plates, broth cultures) TSA+ plates, PCA plates Reagents for Gram reaction Reagents for oxidase and catalase test Microscope

Culturing Techniques: Sterile inoculating loops were used to make streak plates of each strain on TSA+ plates. Incubated at 30 C for 1-3 days

Microscopy: Wet mount was prepared and the morphological identification (shape) was done for the given microorganisms using microscopy.

Gram reaction: Three methods were used for Gram reaction: conventional staining (bright field microscopy), KOH method, and aminopeptidase method.

Enzyme reactions : oxidase and catalase

Results:

Strain number 253 270 230 245 238 311 Rod Oval Rod Rod

Shape

Gram reaction Gram Negative Gram Positive Gram Positive Gram Positive Gram Negative Gram Positive

Oxidase + -

Catalase + + + + +

Family/Genus Pseudomonas Yeast Bacillus Lactobacillus Enterobacteriaceae? Staphylococcus

Coccus Coccus

Discussion and conclusions: Our group was not able to identify strain 238. It is a G- coccus, which is a very uncommon combination (most cocci are G+). It is possible that it was originally a rod, but since it was not a fresh culture when we examined it (4 days old) and it was kept at room temperature, the conditions (T, humidity, lack of some nutrients) might have been inadequate for that microorganism, which can sometimes lead to the change in shape, in this case from rod to coccus. If we suppose that this is indeed what happened, we can identify strain 238 as Enterobacteriaceae, since Gram and enzyme reactions correspond to this group. The other possible explanation is that our plate might have got contaminated when we were making streak plates, so we did not obtain pure culture. Results of groups 21-40, showing correctly and falsely indentified groups of m.o. in percents: %correct
Bacilles Entero Lacto Pseu Staph Yeast 65 63.16 77.27 80.95 90 90.48

%false
35 36.84 22.73 19.05 10 9.52

Next graph\table shows which groups were confused for which, expressed as a % of the total number of mistakes. For example, when the mistake was made in identification of Staphilococcus, in 50% of cases it was identified as a Bacillus and in other 50% as Enterobacteriaceae.

Microorganisms that were reported as detected Bacillus Microorganisms that were supposed to be detected Bacillus Entero Lacto Pseudo Staph Yeast
0.00% 42.86% 0.00% 50.00% 50.00% 100.00%

Entero
28.57% 0.00% 80.00% 25.00% 50.00% 0.00%

Lacto
14.29% 14.29% 0.00% 0.00% 0.00% 0.00%

Pseudo
14.29% 14.29% 20.00% 0.00% 0.00% 0.00%

Staph
14.29% 28.57% 0.00% 25.00% 0.00% 0.00%

Yeast
28.57% 0.00% 0.00% 0.00% 0.00% 0.00%

The following figure shows this data in a graphical way:

100.00% 90.00% 80.00% 70.00% 60.00% [%] 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% Bacillus Entero Lacto Pseudo Staph Yeast Microorganisms that were supposed to be found Bacillus Entero Lacto Pseudo Staph Yeast

Yeasts were identified correctly in very high percentage, because they are easy to recognize by their big size and budding shape. In the cases of misidentification, they were confused with Bacillus, which is not strange, since yeasts and Bacillus are both G+ and give the same results in enzyme reactions, so it is only possible to distinguish them by microscopy.

Most mistakes were made with Enterobacteriaceae, which were commonly mistaken for Bacilus, because they have the same shape(rods), and can have the same results in enzyme reactions ( catalase + for both, and for oxidase Enteros are-, whereas Bacillus can be both + and -). They can be distinguished by Gram reaction, but if only KOH method is used, which is not very accurate, and the results of which can be easily misinterpreted, especially by inexperienced students, the mistakes can easily be made.

Answers: 1. Our results correspond with the given names, except for the strain 238. Explanation is given in discussion and conclusion section. 2. Microscopy can be difficult for inexperienced people, because microorganisms are often clustered in groups and very small, which makes it difficult to determine their shape. What is more, rods can sometimes be very short, and look like cocci. Gram staining consists of several steps, so it is easy to make a mistake, especially with preparation of the smear (too much material) and with heat fixing (if too short- smears tend to be washed away during staining and washing; if too long- staining artefacts, disruption of normal morphology of bacteria might occur). KOH method is very fast and easy to perform, but determination of results can be challenging for an inexperienced person since it is not always easy to determine whether there are threads or not. 3. Conventional staining is time consuming and mistakes can be easily made since it consists of many steps. On the other hand, when obtained, results are very clear. KOH method is fast and easy to perform, but not very accurate and reliable. Sometimes it is hard to determine the presence/absence of threads. Aminopeptidase method is time consuming, but it is very easy to perform and to read the results, even for an inexperienced person. This method is preferable for determining Gram reaction.