Bacterial Growth Analysis: Optical Density

1. To measure the absorbance of the bacterial solution at 600nm

2. To investigate microbial growth

3. To stain the microbe under culture using Gram stain

Introduction

Growth is the increase in the number of the components of an organism. Growth is measured using cell concentration or biomass density. Turbidity, a type of cell concentration techniques, is measured as the absorbance of light at a defined wavelength. Turbidity follows Beer-Lambert’s law which states that molar absorptivity is directly proportional to the concentration of the substance and the path length of the light through the solution expressed as:

A=ebc

Where A= Absorbance and has no units

e is the molar absorptivity with units of L mol-1 cm-1

b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained expressed in centimeters.

c is the concentration of the compound in solution, expressed in mol L-1 (Shefield Hallam University).

Spectrophotometry is a quantitative method used to measure the amount of light absorbed by a substance as a beam of light passes through the sample solution. Spectrophotometry in the ultraviolet range (185-400nm) or visible range (400-700nm) uses a UV-Vis spectrophotometer. Visible spectrophotometers use prisms to filter out unwanted wavelengths such that only a particular beam passes through.

Figure 1 Spectrophotometer Principle

Turbidity can also be visually estimated where a barely turbid suspension is estimated to have 107 cells per milliliter while a fairly turbid suspension has 108 cells per milliliter (Brooks , Carroll and Butel 55). Turbidity is related to viable cell count by a standard curve. Growth parameters are estimated based on either fitting primary growth equations to absorbance data directly, to log-transformed data, or to detection times of serially diluted cultures such as Baranyi-Pin method and decimal dilution method (Lindqvist 4862). The experimental data obtained from each method is usually compared to growth models that are either mathematical or mechanistically derived data known as the maximum specific growth rate (μ max).  

 Growth parameter estimates have classically been obtained from plate counts where serially diluted bacterial cultures are streaked on agar plates and incubated at optimum growth environments. However, his method does not provide precise and accurate date hence the need for automated methods. Turbidity measurements are rapid, nondestructive and relatively cheaper. The limitations to this method include being limited to liquid samples and having high detection limit ranges of 106 to 107 cells ml-1 consequently yielding little information in lag phase

Bacteria are invisible under a microscope and can only be visualized when color is added either to the background or on the cell itself. Staining is a chemical or physical union of a dye and it’s unlike component in a cell. Gram stain is a differential microbial stain that differentiates bacteria into two categories based on the lipid content of the cell wall. Crystal violet dissociates into its constituent radicles which penetrate the cell wall and membrane of both Gram Positive and Gram-negative bacteria and interact with negatively charged cell components. Iodine acts as a mordant crosslinking the inner and outer layers. The decolorizer (alcohol or acetone) interacts with the lipid membrane. The decolorizer causes crosslinking or precipitation of the cell membrane of Gram Negative bacteria and therefore the color is retained. Gram-negative bacteria have a thin lipid membrane which gets dissolved hence decolrized and the stain washed away. Gram-negative bacteria interact with the positively charged counterstain safranin and take up its color while Gram-positive bacteria resist it. When stained with a primary stain and fixed with a mordant, some bacteria are able to retain the primary stain by resisting decolorization while others get decolorized by a decolorizer.  Gram-positive bacteria retain the primary stain (blue/purple) while Gram-negative bacteria get decolorized and take the color of the counterstain which is pink/red (Gram Staining: Principle, Procedure, Interpretation and Animation). 

Materials and Equipment:

1. Growth agar plate 

2. Bacterial suspension

3. Spectrophotometer

4. Cuvette(s)

5. Pipette

6. Gram stain; Crystal Violet, Methylene Blue, Gram’s Iodine and Safranin

Methodology:

Microbial Growth Analysis: Absorbance/Optical density

i. Obtain a bacterial culture previously prepared and incubated at 370C.

ii. Insert cuvette into the spectrophotometer and read absorbance at 600nm then zero the machine.

iii. After every 15minutes insert the cuvette into the spectrophotometer and read off absorbance at 600nm.

Microbial Growth Analysis: Plate Counting (Colony Forming Unit)

i. Set up 6 tubes with 0.9ml of the media

ii. Using a pipette with a sterile tip, transfer 0.1ml of the culture in the tube with 10-1

and mix well. Discard the tip.

iii. Using a pipette with a sterile tip, transfer 0.1ml of the culture to the tube with 10-2

and proceed until the one with 10-6.

iv. Using a pipette with a sterile tip, starting with the highest dilution take 0.1ml of the solution and transfer to an agar plate.

v. Using a splendor, spread out the solution on the surface of the agar plate.

vi. Air dry the agar plate then incubate at 370C.

Gram stain

i. Take a grease-free dry slide.

ii. Sterilize the inoculating loop on a flame of a Bunsen burner.

iii. Transfer a loopful of culture by a sterile loop and make a smear at the center. 

iv. Allow the smear to dry in the air.

v. Fix the dry smear by passing the slide 3-4 times through the flame quickly with the smear side facing up.

vi. Cover the smear with crystal violet stain and leave for 1 minute. Wash with tap water

vii. Flood the smear with Methylene Blue working solution for 1 minute. Rinse with tap water.

viii. Flood the smear with Gram’s iodine solution and leave for 1 minute. Wash with tap water.

ix. Flood the slide with the decolorizing agent then wait for 20-30 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent running from the slides runs clear. Gently wash and drain completely.

x. Counterstain with safranin for 1 minute.

xi. Wash, remove excess water with a blotting paper then air dry.

xii. Observe under a microscope.

Results:

Table 1 Optical Density at 690nm

Time

Sample

OD (690nm)

Number of Bacteria

# of Bacteria (log)

22:47:28

B

0.000

0

0

22:47:29

1

1.724

22:47:30

2

1.383

22:47:31

3

1.041

22:47:31

4

0.738

22:47:32

5

0.404

Table 2 Microbial Growth Analysis (Plate Counting: Colony Forming Unit)

Plate #

Dilution Rate

CFU

Number of Bacteria

Conc. of Bacteria (#/ml)

1

10-1

2

10-2

3

10-3

4

10-4

5

10-5

222

6

10-6

178

Figure 2a) Gram Positive Bacteria b) Gram Negative Bacteria

Discussion

Growth parameters estimates analyzed by turbidity measurements and viable cell count most often than not tend to be equal or with a slight variation. The variation is caused by the selected growth model, nonlinear relationship between absorbance and concentration of the bacterial suspension and using cells that are in the declining phases of growth. Growth parameters obtained from plate counting are more reliable however at low cell densities absorbance estimates are more valid since the latter is an easier process and can be automated (Perni , Andrew and Shama ). There is no criteria in choosing the growth model and it is dependent on a trial and error basis. The experiments are done in duplicates to increase accuracy and the values obtained are usually an average.

Conclusion

Turbidity methods offer rapid and reproducible estimates of growth parameters. However, the results are compared to the classical viable cell count for accuracy. The two methods are related by a standard curve or by comparing the results to growth models. 

Questions.

1. Why should the absorbance of the analyte not be too high? Absorbance values are dependent on the wavelength used. When absorbance is too high, it will require a higher wavelength and therefore will cause a shift from the visible wavelength in the spectra, the spectrum utilized in spectrophotometry.

2. What is the reason for measuring absorbance at 600nm in the experiment? Bacterial cells are transparent and will scatter too much light leading to a reduction in absorbance. Therefore bacterial growth cultures are used in turbidity measurements. The bacteriological growth media contain substances which tend to have a higher absorbance at 400nm but have normalized absorbance towards the blue end of the visible spectrum which is 600nm.

3. Describe each phase of the microbial growth curve.

Lag phase. This phase is marked by zero growth rate as the bacteria have depleted the enzymes and metabolites they had acquired in a previous culture. Growth resumes once metabolites and enzymes have accumulated to optimum levels. If bacteria are transferred to a new medium while in this phase, the probability of death is high.

Log phase or exponential phase.

There is a constant growth rate where new material is synthesized at a constant rate however the material is also catalytic consequently an exponential increase in mass. This is altered when either one or more growth factors are eliminated or toxic metabolites accumulate and inhibit growth.

Stationary phase. There is zero growth rate caused by inhibited growth and cell turnover. Cell death is minimal however it is balanced by cell growth and division.

Decline or death phase.

There is a negative growth rate. Death rate increases until it reaches a constant rate. After most cells have died, the few surviving cells may persist for long presumed to be feeding on the materials from the dying cells. Therefore, the curve does not return to zero.  

4. Explain the role of each component used in serial dilution.

i. Stock solution. It is the original concentrated solution that is taken through steps to obtain a diluted solution.

ii. Test tube. It holds the sample that is being diluted.

iii. Pipette. Measure and transfer exact volumes of solutions.

iv. Water or any other solvent. It tops up the volume to the original volume after the diluted sample has been transferred.

Works Cited

Brooks , Geo F, et al. Jawetz, Melnick, & Adelberg’s Medical Microbiology. The McGraw-Hill Companies, 2013.

Gram Staining: Principle, Procedure, Interpretation and Animation. 19 January 2016. 17 October 2018. .

Lindqvist , R. "Estimation of Staphylococcus aureus Growth Parameters from Turbidity Data: Characterization of Strain Variation and Comparison of Methods." American Society for Microbiology July 2006: 4862-4870. https://aem.asm.org/content/aem/72/7/4862.full.pdf.

Perni , Stefano, Peter W Andrew and Gilbert Shama . "Estimating the Maximum Specific Growth Rate from Microbial Growth Curves: Definition is Everything." Food Microbiology (2005): 491-495. http://www.sciencedirect.com/science/journal/07400020.

Shefield Hallam University. n.d. https://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/beers1.htm. 22 October 2018. .