Preparation of McFarland Standards

 

Purpose:

 McFarland Standards are turbidity standards that are used to gauge approximately how many bacteria are present in a liquid suspension. The standards are used to visually compare the turbidity of a Bacterial suspension with the turbidity of the appropriate standard.  Standards are prepared by adding barium chloride to sulfuric acid to obtain a barium precipitate. The volumes of the two reagents are adjusted to prepare standards of different turbidity that represent different concentrations of bacteria.

 

Reagents:

1.       Sulfuric acid, 1%

2.       Barium Chloride, 1.175%

 

Supplies:

1.       Acid washed glass screw-cap tubes comparable to that used for test

2.       Sterile serological pipettes and pipette bulb

3.       Parafilm or paraffin

 

Equipment:

1.       100ml volumetric flasks

2.       Vortex

3.       Spectrophotometer

4.       Magnetic stirrer and stirring rod

 

Procedure for the Preparation of a 0.5 McFarland Standard:

1.       Add approximately 85 ml of 1% sulfuric acid (H₂SO₄) to a 100ml volumetric flask.

2.       Using a volumetric pipette, add 0.5ml of 1.175% anhydrous barium chloride (BaCl₂) drop wise to the 1% sulfuric acid (H₂SO₄) while constantly swirling the flask.

3.       Bring the volume to 100ml with 1% H₂SO₄.

4.       Stir or mix for approximately 3 to 5 minutes while examining visually, until the solution appears homogeneous and free of clumps. A magnetic stirrer can be used for this step if available.

5.       Check optical density, following the procedure described in the QC section below and record on QC sheet.

6.       If QC is acceptable, dispense 2 to 7 ml volumes (depending on volumes routinely used in test) into each glass screw- cap tube.

7.       Label the tubes appropriately including the expiration date and the initials of the person preparing the standards. Make sure that the labeling is positioned so that it does not interfere with spectrophotometer readings.

8.       Cap the tubes tightly.

9.       Draw a line to mark the meniscus on each tube. This mark can be used as a guide to check for evaporation at a later time.

10.   Seal the tubes with paraffin or Parafilm.

11.   Repeat the procedure to make additional standards using volumes indicated in Appendix A.

12.   Store the prepared standards in the dark at room temperature for 3 months or longer as per QC acceptability.

 

QC:

1.       At time of preparation check the optical density (OD) of the McFarland standard at a wavelength of 625nm and record results. The acceptable range for a McFarland 0.5 standard is                         0.08 to 0.10.OD. For standards other than 0.5 establish acceptable ranges in-house.

2.       Visually check standards with each use for evidence of clumping and/or evaporation. Discard the standard if either is apparent

3.       Check the line drawn to indicate the position of the meniscus during the preparation. If there has been significant evaporation discard the standard.

4.       Check the optical density of a representative standard (that has not been in use) at three months to determine if the optical density is still within limits. If in control, the shelf life can be extended for another month. Repeat check monthly for up to a year.

 

Use of McFarland Standards:

1.       Mix standard and test suspension using a vortex, prior to examination.

2.       With good lighting, visually compare the turbidity of the test suspension to the McFarland standard.

3.        If the suspension is too dense in comparison to the standard dilute the suspension until it is comparable to the McFarland standard.

4.       If the suspension is too dilute in comparison to the standard, inoculate it with additional organism until the concentration matches that of the standard, or prepare a new suspension.

5.       All adjustments to the bacterial suspension should be performed using sterile technique.

6.       A Wickerham Card (see Appendix B) can also be used as an additional guide when adjusting a bacterial suspension to match the appropriate standard.

 

References:

1.       ASM Manual of Clinical Microbiology 2007.

2.       K.C. Chapin and T. Lauderdale. 2003. Reagents, stains, and media: bacteriology, p. 358. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of Clinical Microbiology, 8^th ed. ASM Press, Washington, D.C.

3.       www.google.com

 

Appendices

1.       Appendix A: Guide for the Preparation of McFarland Standards

2.       Appendix B: Wickerham Card Information

 

 

 

 

 

 

Appendix A:  Guide for the Preparation of McFarland Standards

 

Volume in mL
Standard	1% BaCL2	1% H2SO4	Number of Bacteria/ mL/(108) represented
0.5	0.5	99.5	1.5
1	1.0	99.0	3
2	2.0	98.0	6
3	3.0	97.0	9
4	4.0	96.0	12
5	5.0	95.0	15
6	6.0	94.0	18
7	7.0	93.0	21
8	8.0	92.0	24
9	9.0	91.0	27
10	10.0	90.0	30

.

Serial Dilution and counting method for microorganism:

A serial dilution is a process were the concentration decreases by the same quantity in each successive step and concentration of microorganism in different dilution factor. We used three methods in microbiology for enumeration of microorganisms or counting of microorganisms. Pour plate method, direct microscopic count method, spread plate method. In all three methods described in lab require to serially dilute sample until you have 30-300 colony forming units (CFU) on the plate.  Plates which contain more than 300 CFU are very difficult to count. And plates contain less than 30 CFU are not statistically reliable.

There are some diluents used in microbiology which are given below:

·         Saline water.

·         Peptone saline diluents.

·         Buffered peptone water.

·         Sodium citrate diluents.

·         Di potassium hydrogen phosphate diluents.

 

Method for Dilution:

·         Prepare Diluents transfer 9 ml diluents into each tube and Label all diluents blank as  10^-1, 10-², 10^-3, 10^-4, 10^-5, 10^-6etc.

·         Prepare initial dilution adding 10gm sample + 90 mL diluent or 25 gm sample + 90 mL diluent mix by vortex to obtain uniform distribution of microbes.

·         From first dilution using a sterile 1mL pipette, transfer 1mL of suspension into 10^-1 Test tube

·         And transfer same 10^-1 to 10^-2, 10^-2 to 10^-3, 10^-3 to 10^-4 and up to 10^-6.

·         Repeat this procedure till the original sample has been diluted up to 10, 000, 00 (10^-6) times using every time a fresh sterile pipette tip.

·         After preparing dilution liable Petri plate 10^-1 to 10^-6, 1mL suspension of each dilution tube transfer into each dilution liable sterile glass or plastic Petri plate.

·         Add approximately 15 ml of the palate count agar or Nutrient agar medium, melted and cooled to 45⁰ C, to each Petri plate containing the diluted sample.

·         Mix the content clock wise and anti -clock wise and incubate appropriate temperature for selective time.

·         Observation: Observed all plate for the appearance of colony.

·         Count number of colonies in the Petriplates that have colonies in the range 30-300 CFU range count plate by colony counter.

 

Calculation: according to Indian standard-

                      

 
Where

ΣC = the sum of the colonies counted an all the plates;

n₁=the number of plated counted in the forts dilution;

n₂ = the number of plates counted in the second dilution

D= the dilution from which the first counts were obtained (for example,10^-1)

 

 

For Example:

10^-3 dilution: 83 and 97 Colonies

10^-4 dilution: 33 and 28 Colonies

                                           =10954 CFU /gm or 10954 CFU/ml    

References: IS 5403 (1999): Method for Yeast and Mould Count of. Foodstuffs and animal feeds.

       Experiments in Microbiology, Plant Pathology and Biotechnology by K.R. Aneja

                    www.google.com

                    

Good microbiological laboratory practice:

·        Always wear a laboratory coat or apron before entering a laboratory for protecting clothes from contamination or accidental discoloration by staining solution.

·        Wearing appropriate garments (e.g. gloves and shoe covers or lab shoes) when enter into laboratory.

·        Never eat, drink, apply cosmetics or lip balm, handle contact lenses or take medication in the laboratory.

·        Never wear jewelry (e.g. Watch, ring, etc) in microbiology laboratory.

·        Use aseptic techniques. Hand washing is essential after removing gloves and other personnel protective equipment, after handling hand sanitize with 70% IPA or 90% ethanol.

·        Microbiological testing should be performed and supervised by an experienced person, qualified in microbiology or equivalent. Staff should have basic training in microbiology and relevant practical experience before being allowed to perform work to pathogens.

·        Microbiology laboratories and certain support equipment (e.g. autoclaves and glassware) should be dedicated and separated from other areas, especially from seating areas.

·        Before and after each laboratory period, clean your workbench with a disinfectant like 70% IPA.

·        Keep your laboratory bench clean or everything (books, purses, paper, etc) except your laboratory equipment and notebook.

·        If a live culture is spilled, cover the area with a disinfectant such as mercuric chloride for 15 minutes and then clean it.

·        In the event of personal injury such as cut or burn, inform your instructor immediately as bacteria love open wounds.

·        Wash your hands with soap and water before and after finishing work in the laboratory.

·        Broth cultures must never be pipette with mouth and must be filled with the use of pipette aids and operated in such a way so as to avoid creating aerosol

·        Aseptic technique must be rigorously observed at all times.

·        Always keep culture tube in an upright position in rack or basket.

·        You must familiarize yourself in advance with the exercise to be performed.

·        Materials such as stain and reagent bottles, Petri plates, pipettes must be returned to their original location after used.

·        Always clean microscope stage, eyepiece, lenses and objectives, before and after use. All lenses must be wiped with the xylene or lens paper.

·        Keep the laboratory doors and windows closed when experiments are in progress. Precaution must be taken while removing the closures from shaken culture tubes and plunging of contamination loops into a flame to avoid inhalation of an infectious microbe, in other words, to avoid infection from laboratory.

·        All instruments like incubator, autoclave, hot air oven, laminar air flow, Bio safety cabinet etc internally calibrate on stable time duration.

·        Keep your lab logbook up-to-date.

Escherichi coli

Scientific classification

Domain:	Bacteria
Phylum:	Proteobacteria
Class:	Gammaproteobacteria
Order:	Enterobacteriales
Family:	Enterobacteriaceae
Genus:	Escherichia
Species:	E. coli

 

Escherichi coli is a normal inhabitant of the human digestive tract, some E.coli caused iarrheae diseases in humans.

Most E. coli are harmless and actually are an important part of a healthy human intestinal tract. However, some E. coli are pathogenic, meaning they can cause illness, either diarrhea or illness outside of the intestinal tract. The types of E. coli that can cause diarrhea can be transmitted through contaminated water or food, or through contact with animals or persons

These pathogenic E.coli are classed nto five groups:-

• ·         Enterotoxigenic (ETEC)
• 
  
• ·         Enterohemorrhagic (EHEC)
• 
  
• ·         Enteroinvasive (EIEC)
• 
  
• ·         Enteropathogenic (EPEC)
• 
  
• ·         Enteroadherent-aggregative (EA-AggEC) new Reconized Group. This is aggregative or “Stacked-brick” like aherence to cultured mammalian cells.

Pathogenic E.coli can be devided on the basis of serology but, because they are classedon the basis of pathogenic factors.