Quantitative Enumeration of Microorganisms & Factors Influencing Microbial Growth

Introduction

Microbiologists determine the number of microorganism in a sample given and as well compare it to numerous conditions. Such as enumeration of microorganisms. This is extremely significant in microbiology, water and food microbiology (Montville & Matthews, 2008). The process of quantitative enumeration is to regularly use to help many laboratory microbiologists to see the growth of unicellular microorganism such as bacteria, its reproduction of cell reproduces the entire organism (Kramer & Gilbert, 1978). This will determine rates of microbial growth and also death and it’s essential for scientists to enumerate microorganisms, to determine the number of individual viable in a sample (Alderson,2011). There are several methods that can also be used for microorganism enumeration such as, pour plating, spread plating, swab count, turbidity measurements, counting chambers and involves experiments from serial dilution to plating of aliquots in a medium to detect colonies. Microbiologist uses the term colony-forming-units in quantitative plate count (Cowan & Smith, 2018). All of these methods help to distinguish different microorganisms accurately. The requirements for microbial growth can be divided into two main categories: physical and chemical. Physical aspects involve temperature, osmotic pressure and pH. As for chemical aspects, the requirements require sources of nitrogen, carbon, sulfur, phosphous, oxygen and organic growth factors. Microorganism grows well at the temperature that human favours. Some bacteria are capable of growing at extreme temperature and some bacteria are capable to grow in lower temperatures. The microorganism is classified into three groups: psychrophiles, mesophiles and thermophiles (Montville & Matthews, 2018). Microbiologist must deal with microorganisms which can be affected by their external environment and internal factors which is significant in microbial cell growth and reproduction (asexual). In the experiment, different factors such as temperature, pH, osmotic pressure, metals will have an impact on the microorganisms observed.

This lab report shows the different factors such as oxygen, temperature, pH, osmotic pressure and metals can affect microbial growth. The microorganism can grow under aerobic or anaerobic conditions. By placing the same microbes in different temperatures, we can see the range the microbe can grow in along with hat is considered the optimal temperature. As for pH levels, it will be determined what the optimal pH level is for growth, along with the range in which growth is possible. Lastly, we consider the effects of osmotic pressure. This indicates if the cell is hypertonic, isotonic or hypotonic. The objective of quantitative enumeration of microorganisms is to examine different types of quantitative enumeration and to perform quantitative enumeration of living organisms. The objectives for factors affecting microbial growth is to determine growth characteristics of different temperatures, determine the responses bacteria and fungi of solute (concentration), demonstrate the effects of metals on certain bacteria and compare fungal growth with bacteria growth at different pH levels.

Method for Quantitative enumeration of microorganisms

Take 4 tubes of 9ml peptone water. Label tubes with dilution (10^{-1 ,}10^{-2 ,}10^-3 ,10^-4). prepared the 10^-1dillution, pipette 1ml of the tube of S. cerevisiae. Transferred this to your 9ml peptone water labelled 10^-1. Strictly flame the mouths of tubes. Shake the tube well using a vortex mixer or rolling the tube between palms of your hand. Used steriled pipette, transfer 1ml aliquot from the 10^-1 dilution into the 9ml tube and shake well.

Pour plating

Take 4 sterilised Petri plates and label it as -1,-2,-3,-4.

Take 1ml of aliquot from each dilution into the appropriate plate. Make sure a fresh sterile pipette tip should be used to take out a sample from each dilution.

Poured 15-20 ml of molten agar into each plate. Be sufficiently quick to avoid the agar solidifying before it is mixed with the sample.

Mix carefully

Spread plating

take 4 plates of pre-poured PCA

transfer 0.1ml aliquot from each plate

flame sterilize a glass spreader by immersing in ethanol and quickly burning in a Bunsen flame. Use this to spread the aliquot on the surface of the agar. Make sure agar is not broken.

Swab count

1. Choose an area which you are going to examine
2. opened the swab container and removed a swab
3. opened the bottle of peptone water containing glass beads. Immersed the swab into the solution.
4. Pressed out the excess fluid on the neck of the bottle
5. Opened the container of template and remove the template with flame sterilized forceps.
6. Rubbed the swab over the whole of the exposed area of the template. Rotated the swab head while rubbing the area. Rubbed the swab up and down, at right angle and at 45-degree angle.
7. Return the swab into the bottle of peptone water. Carefully break the stick between 2-4 cm of the handle remain attached to the head.
8. Closed the bottle. Shake vigorously 5-10 times
9. Used a sterile pipette tip and transfer 0.1ml of the suspension into each of two Petri dishes. Then, transfer 1.0ml each into another two petri dishes.
10. Covered with molten agar and mixed well. Allow the set and incubate until colonies are visible.

Turbidity measurement

1. Measured the turbidity of the suspension by using a spectrophotometer. This will measure the amount of light that passed through the liquid medium.

Method for factors influencing microbial growth

Gathered 3 plates of NA. Marked the bottom of the dish into 5 equal sections and label each plate with the E. coli, S. marcescens, and the last plate with B. subtilis. Sterilized a pair of forceps using alcohol and Bunsen flame. Used the forceps to pick up a sterile filter paper disc from the container. Immersed the disc into the solution of metal. Deposited the disc carefully onto the appropriate section of the agar. Incubated plates at 30 degrees until colonies were visible.

Experiments for other factors influencing microbial growth were arrived out by other groups

Results

Effect of temperature on growth

Effect of pH

NaCl concentration (%)

Glucose concentration (%)

Effect of metals

Quantitative enumeration of microorganism’s results

Viable plate count: pour plate:

Viable plate count: spread plate

Swab count:

Turbidity Measurement

Discussion

As for quantitative enumeration of organisms, it requires good practices and going through the technique carefully as a group. This is will give good results to be used for the calculations and also providing accurate results. As a group, we’ve carried out good enumeration and aseptic techniques. During the quantitative enumeration of pour plating, the group followed the lab manual instructions effectively. As for the swab count experiment, it was done falsely because the swab was rubbed over the unprotected area of the template but then was thrown away into a container with other contaminant plates. Therefore, this has led to incorrect results of microorganisms. Another mistake was the effect of metals, the experiment was incorrectly done due to extra metal solutions. Thus, these experiments must be handled with extreme caution to prevent any necessary inaccuracies in the future.

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The advantages and disadvantages of quantitative enumeration are the viable cell counts of spread and pour plating, an advantage of this technique is the measurement of live cells growing on the plate. As for disadvantages, is not measuring dead cells because necessary time will be wasted. This is significant because it limited microorganisms, as they only know how to grow (Alderson, 2011). For turbidity measurement, an advantage of measuring cell density and shows instantaneous microbial growth are ideal (Alderson, 2011). A disadvantage is when this technique needed a high concentration of cells but the spectrophotometer cannot differentiate between dead or alive cells (Alderson, 2011). Lastly, the serial dilutions, the advantage is to decrease the dense culture of cells to a more fitting concentration and a specific number of microorganisms are decreased with every dilution (Alderson, 2011).

Factors influencing microbial growth can occur in numerous ways. First of all, the temperature effects have impacted three organisms of Escherichia coli, Penicillium chrysogenum and Pseudomanas aeruginosa. These three organisms are mesophie (moderate-loving microbes), that grows between 25°C and 40°C, optimum temperature commonly 37 degrees (Cowan &Smith,2018). E. coli organism grows over temperatures around 40 degrees and the cell rate increases around 20 degrees to 37 degrees. As for mould organisms such as P. chryosogenum, the optimum temperature is 23 degrees and 28 degrees (Dale, 2012).

The effect of pH levels can have a significant impact on microbial growth. Bacteria favours conditions between pH of 6.5 and 7.5. Fungi grows best at lower pH levels between 5 to 6.5 (Dale, 2012). Moulds and yeasts can grow in more acidic conditions and bacteria can grow in more base conditions.

Metals is vital to cell functions of microorganisms in lesser concentrations, this is referred to micro-nutrients (Cowan & Smith,2018). Extreme concentrations of some metals can inhibit actions against organisms, this could be toxic to microbial cells. The percentage of certain mental (copper or mercury) harming cell of microbial is called oligo dynamic action (Cowan & Smith, 2018). Also, the extreme concentration of a few metals can inhibit actions against microorganism and is toxic to microbial cells. However, the only metal to create an inhibition zone in all three of our microorganisms is copper. Copper contains antimicrobial properties wherein oxic conditions, copper catalyses a Fenton-like reaction. This causes damage to lipid peroxidation and protein by shattering the cell membrane. Therefore, this lead to interrupting osmotic pressure when microorganism is near copper mental (Cowan & Smith, 2018).

Conclusion

Microbiologists determine the number of microorganism in a sample given and as well compare it to numerous conditions. As for microbial growth, a plate is used to determine the number of microorganism living in a sample.

References

• Cowan, M. K., & Smith, H. (2018). Microbiology: a systems approach. McDraw-Hill Education.
• Dale, J. W. (2012). Understanding microbes: An introduction to a small world.
• Kavanagh, K. (2018). Fungi: biology and applications. Wiley Blackwell.
• Montville, T.J., & Matthews, K. R. (2018). Food microbiology: an introduction, ASM
• Alderson, G.D. (2011). Microbiology: experiments & lab techniques. Fountainhead.