Growth of Microbes Using Hay Infusion Experiment

• Jekathjenani Ratnakumaran

Lab # 6

Hay Infusion

Introduction:

The micro organisms grow in everywhere on the earth. They can be found in the air, water, and the earth’s surface. They need only the growing factors. Growth factors are varying from microbes. Most of the bacteria grow in the environment with the moderate temperature, neutral pH, and the sufficient O₂ supply. Some of them can live in the very high temperature and some can live in very cold temperature. Most of the bacteria can live in the oxygen environment and are called aerobic bacteria and some of the bacteria that exist without oxygen called anaerobic bacteria. The cyano bacteria bring the oxygen to the air. Most of the bacteria help in the decomposition of dead animal body and plant body. The microbes help in food preservation.

The purpose of this study is to learn about the growth of microbes using the hay infusion method. The hay infusion is the culture made in the laboratory with the water collected from the ponds, and lakes. This hay infusion will allow viewing the microbes developments over the time period and it is a great way to produce the different kind of organisms around the time period. Microbes are very small, therefore it is very difficult to see through the human eye. This hay fusion experiment allows seeing the microbes through the microscope via wet mount. This experiment was run through the six weeks of the lab. First day of the lab was only preparing the hay infusion culture with the given materials. The hay infusion made using the mud sample which was collected around the Trent university area. The sugars in the dried grasses provided for the food source for the microbes. The hypothesis of the study, an increase in the availability of more diversity of microbes at the bottom layer of the hay infusion beaker. This hypothesis can be tested by predicting that the bottom layer has more nutrition and more mortality than other top and middle layers.The microbes live in the more nutrient environment. They would like to hide their self from the light source and they can obtain more stable motility around the bottom layer. Therefore, these microbes are favored to live at the bottom of the beaker.

Method:

In the first week of the study, firstly 1500ml of beaker was taken and few amount of hay was placed in the beaker and filled with enough amount of water to cover the hay in the beaker. The beaker with the content was placed in the heat source for 30mintes. After that the hay with the beaker was cooled down to room temperature for an hour. The same quantity of river water was put in the hay mixture and kept the 5 cm space left from the upper side of the beaker. The small amount of hay was left in the beaker as it was enough to form the loose mass and light passes through the layer in the bottom of the beaker. Later, the two tablespoons of mud added to the beaker along with the hay and the cooled water. Then the beaker was carefully covered with the tin foil paper and was ensured that the side of the beaker, not covered because the light has to pass through the beaker to provide the growth environment for the microbes. The beaker was placed in the environmental chamber.

From the first day of the preparation of hay infusion and continued for six weeks to observe the microbes formation and development in the hay fusion culture. The beaker was divided into three portions as a top layer, middle layer and the bottom layer. The beaker was collected from each week and placed on the lab table without mixing the content. The three labelled plastic pipette was used to collect the drop of the sample from the top layer and placed in the slide, covered with the cover slide and was examined under the light microscope to identify the microbes.

Results and Observations:

Week 1: There were no microbes was detected as the study’s method include only for the preparation of the hay infusion.

Week 2:

Top layer of the beaker has some dark color, small pieces were hung up from the surface of the layer. There was no change in the water level and it was remained same as before like 1000ml. The beaker was analyzed in the room temperature. There were some organism was found with single layer of body structure with the nucleolus in the middle of the body. It was identified as paramecium, called Frontonia. It had cilia around the body. The cilia structure was indicated that microbes able to do motility. The body shape was a rod like shape and light brown in color. This organism was named with the aid of a guide book. It had very fast movement.

The middle layer was little cloudy and couldn’t find any microbes moving around the layer by seeing with the naked eye, however the wet mount was created by the drop of sample from the middle layer and viewed under the light microscope, there only one organism called Synedra found. They showed the little motility and they were in rod shape. They showed no movement.

The bottom layer was looking very dark color. The bottom layer was found as very brown color. Also, there was no any visible organisms moved around by observing through the naked eye. However, the wet mount was created and watch those three layers under the light microscope some of the organisms were detected. The wet mount was created for the bottom layer and looked under the light microscope. There were lots of microbes were detected. Paramecium was commonly detected all over the slide surface. They were bigger in body size and had high motility. And some of the protozoan also identified with the high movement. The green color organisms also identified it was cuboid in shape. It had a bunch of rod shaped clusters joined together and called Fragilaria. Also, other organism which is large, long shaped, clear colored was found and it’s called Nitzschia.

Week 3:

The top layer surface of the layer looked like the very dark color; dark black color film was growing up. Some air bubbles were stuck on the side of the beaker and the edge of the beaker. The water level was at the same level as before week. The beaker was observed in the room temperature. There were no visible things moving around the top layer while looking with naked eyes. The wet mount was created and looked under the light microscope, there were only microorganisms were found was Paramesium.

The middle layer looked little clearer than the last week. Some small things moving around the middle layer while looking under naked eye. Those small things looked alike hay showed swimming movement in the middle layer, however, those swimming things were not an organism and they were the part of the grass, which used in the preparation of the hay fusion.

The bottom layer was in dark colors as last week. There were no things moving around the bottom part of the beaker while watching with the naked eye. The organisms that are called was Pinnularia found. It was linear in form. Its body was segmented horizontally and the front face has two dark spots. They were ladder like shape. They were in green color and they were not moving. Paramecium was found in the bottom layer and had very fast movement compared to other organism.

Week 4.

The top layer was observed and the water level was going down. The level was moved from 1000ml to 800ml. A lot of air bubbles were hung on the side of the beaker. The dark brown color molds were developed in the surface of the top layer. There were no organisms observed in the top layer while looking with the naked eye. The wet mount was created and looked under the light microscope there were lots of paramecium were moving around.

The middle layer looked clearer than the previous weeks. It’s seems in green color. The paramecium was found under the light microscope. An organism with an oval shaped body and had inner bubbles like structure, it’s called Campylodiscus were observed.

The bottom layer was darker than previous weeks. The volume of the water level was lowered. There were small things moving around the bottom of the beaker. The wet mount was created and looked under the light microscope, there were lots of small things were crawling. The organism called Cymbella was found. Its body was segmented vertically and had branched like body structure and appeared like a leaf structure. It’s non motile organism. Another organism found was called Euchlanis. It was motile, colorless and had antennae and its body was shown squeezing movement. Also, paramecium was found as usual and moved all over.

Week 5:

The top layer was a darker color. The surface of the beaker was covered with the thick black layer of molds. The molds embedded out of the layer. They smell very bad. There were too many bubbles were obtained on the surface of the layer and the side of the beaker. There were no moving things were encountered with naked eyes. The wet mount was observed under the microscope there was an organisms keep on changing the shape of the body. And it was a light green color. It was moving very rapidly. This organism was identified as amoeba. There were lots of Paramecium also identified. There was another microbe called Cymbella also identified which was found already in the fourth week.

The middle layer was green in color. The color of the middle layer was appeared clearer than the previous weeks. There were no small things swimming around the middle layer. Some of the Amoeba was observed under the light microscope. They were very small in their size an appeared light green in color. Synedra was identified as well in the layer. It was a long, cylindrical shape body. The body was segmented vertically.

The bottom layer was dark in color and there were no small things moving in the bottom layer. The parameciums were moving around in the bottom of the layer in large number and in large size when observed under the microscope. Also, Cymbella and already observed diatoms were found in the bottom layer. Another organism called Diatoma was identified. It was appeared rectangular shape with dark spot inside the body. It’s non motile and appeared green in color. Moreover, other two newly identified organisms called Oscillatoria and Dicranophorus. The Oscillatoria was appeared as green in color, non motile and had segmented body. Dicranophorus was appeared as light brown in color, had two antennae in the front part of the body. Also, it had a tail like structure at the posterior part of the body.

Week 6:

The top layer was smelling very bad. The moles were grown outward on the top surface of the beaker. The water level of the beaker was more down (500ml) than the previous weeks. The beaker was obtained warmer than the room temperature. The top layer contains more bioflim. The wet mount was created and looked under the light microscope, there were lots of Amoeba was identified. And they were small in their body size. The oval shaped organisms also detected and it was identified as Trinema. It had a nucleus located in the middle and appeared light green in color.

The middle layer was appeared clearer than the previous weeks. The sample was observed under the light microscope and there were uncountable number of paramecium were identified. The organisms with the rod like shape body and green color pigments inside of the body structure. It has the body structure with the cilia, which appeared around the body and the organisms identified as a Frontonia, a paramecium.

The bottom layer was darker than previous weeks. Parameciums were grown in large number while watching through the light microscope. There were green algae have grown up too. Navicula, Synedra and Nitzschia were observed were identified through the light microscope.

Figure 1 represents the overall number of diversity of organisms present in each layer of hay infusion study. The bottom layer was obtained the highest number of microorganisms compared to top layer and middle layer.

Moreover, the microbial growth was tested by analyzing the samples from each layer using agar plate. The agar plate was observed after it had incubated for 24 hours and the results were recorded. The agar plate had more bacterial growth in the bottom layer compared top and bottom layers. The top layer of the sample had more microbial growth compared to the middle layer. The bacterial growth was slightly raised up from the surface, circular shaped and appeared as solid, creamy in color. They were small in size. More growth was appeared at the edge of the agar plate.

Discussion:

The microbial growth was analyzed and observed using hay infusion method. This study was done to identify the microbe development in the closed system of hay infusion. The six weeks provided to examine the microbial growth and the microbial development in the hay infusion culture. The time period was so short to encounter the microbe’s development. Usually a hay infusion technique carries out the many months to get the microbe development.

The hay infusion provides the food to the microbes via sugar and grass, which added to the hay. The hay infusion may rich in bacterial growth and these are will be the food for protozoan. After several weeks, the protozoan growth was detected in high number. If the protozoan is observed in high number, it means the bacterial growth in the beaker was high too. These bacteria can be harmful to the human and therefore, the students advised to use the gloves while doing the examination of the microbes developments. The water level of the beaker was stated going down for the week of 4. This water level going down will be one of the reasons that microbes were using it as a food source or the water might be evaporate due to the temperature of the lab.

The microbes in the hay infusion may come from the dead grass that added to the beaker. Some of them arrived through the air, which conducted to the surface of the water level. The microbes don’t have the wings, however, these microbes are smaller than the dust, which carry out by the air, so the microbes can easily carry out by the air. The mud which added to the hay infusion culture may contain the eggs of the microbes so they might develop by the time and the environment which provided in the closed system. From the second week of the lab examination until the end of the lab examination of the microbes, the paramecium was commonly detected in highest number. Only one kind of paramecium was detected it might be the reason of the dominants paramecium might be eaten other kinds. The amoeba development was in the last two weeks. The protozoan development was mostly in the bottom of the beaker.

The bad smell was coming out due to the molds formed in the surface of the top layer, and the dead grass parts decomposed with the microbial activity therefore the smells came out. The closure system was used in these experiments, therefore, the oxygen amount in the beaker was reduced by the microbe’s development. There was no other way to give the oxygen supply to the system so most of the microbes were dead in the last week of examination. Also, the light source affects the growth of any organism and therefore, increase in the amount of heat destroy the cell especially, Euglena gracilis (Cramer and Myers, 1952).

The hypothesis was proven by the result of the experiment. The bottom layer has more microbial growth and development. The figure 1 indicated that bottom layer had more diversity of organisms compared to other two layers. Also, microbial growth tested by using agar plate also proven that bottom layer had more bacterial growth compared to other two layers. The top layer was lack of nutrition and not stable environment and bright light passes through the layer. The microbes cannot prefer the top layer because some of the microbes were excited to the light so they tend to hide their self; the bottom layer has lots of layers of hay and dead grass parts which will help to provide the place for them to hide from the light and their parasites. The middle layer was providing the motility space, however, it won’t provide the hiding space and therefore, they might easily caught by their enemies and also the middle layer was poor in nutrients so the microbes prefer the bottom layer to grow and develop. The bottom layer has different kind of organisms due to the layer has more nutrients and stable motility and hiding space and proper intensity of light.

In the future study, hay infusion must be carried out in open system. Hence, it will provide enough oxygen to the system and also, the more microbial development can be noticeable. If the system is close system, then the system should be supplied with oxygen by using appropriate pipette.

References

Jahn, T. L. 1934. Problems of population growth in the Protozoa. Cold Spring Harb Symp Quant Biol 2: 167-180. doi:10.1101/SQB.1934.002.01.022

M. Cramer and J. Myers.1952. Growth and photosynthetic characteristics of euglena gracilis. 17 (4): 384-402

Smolly, C. 2014. Hay Infusion. Biology Laboratory Manual. Department of Biology, Trent University, Peterborough, Ontario