MacConkey agar

MacConkey agar is a culture medium designed to grow Gram-negative bacteria anddifferentiate them for lactose fermentation.

It contains bile salts (to inhibit most Gram-positive bacteria), crystal violet dye (which alsoinhibits certain Gram-positive bacteria), neutral red dye (which stains microbes fermentinglactose), lactose and peptone.

Composition: [Allen, 2013]

Peptone - 17 g Proteose peptone - 3 g Lactose - 10 g Bile salts - 1.5 g Sodium chloride - 5 g Neutral red - 0.03 g Agar - 13.5 g Water - add to make 1 litre; adjust pH to 7.1 +/- 0.2

There are many variations of MacConkey agar depending on the need. If the spreading orswarming of Proteus species is NOT required, sodium chloride is omitted. Crystal violet at aconcentration of 0.0001% (0.001 g per litre) is included when needing to check if Gram-positive bacteria are inhibited.

Mary E. Allen, 2013. MacConkey Agar Plates Protocols. Visual Resources. AmericanSociety for Microbiology, Washington,DC. http://www.microbelibrary.org/index.php/component/resource/laboratory-test/2855-macconkey-agar-plates-protocols (Created: Friday, 30 September 2005, Last update:Monday, 01 April 2013), retrieved on 25/06/2013.

MacConkey Agar Plates Protocol

Mary E. Allen, 2013. MacConkey Agar Plates Protocols. Visual Resources. AmericanSociety for Microbiology, Washington,DC. http://www.microbelibrary.org/index.php/component/resource/laboratory-test/2855-macconkey-agar-plates-protocols (Created: Friday, 30 September 2005, Last update:Monday, 01 April 2013), retrieved on 25/06/2013.

History

MacConkey agar was the first solid differential media to be formulated. It was developed atthe turn of the 20th century by Alfred Theodore MacConkey, M.D, then AssistantBacteriologist to the Royal Commission on Sewage Disposal, in the Thompson-YatesLaboratories of Liverpool University, England. The goal was to formulate a medium thatwould select for the growth of gram-negative microorganisms and inhibit the growth ofgram-positive microorganisms. Dr. MacConkey first developed a bile salt medium containing

glycocholate, lactose and litmus, to be incubated at 22°C (MacConkey, 1900). This formulawas soon altered by the replacement of glycocholate with taurocholate and the incubationtemperature was raised to 42°C (MacConkey, 1901). MacConkey later changed the recipeagain by substituting neutral red for litmus (MacConkey, 1905), following the suggestion thatneutral red be used as an indicator in bile salt lactose medium (Grunbaum and Hume, 1902).The final media formulation was designed to support growth of Shigella and is the one that ismost commonly used today.

Purpose

MacConkey agar is used for the isolation of gram-negative enteric bacteria and thedifferentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria. Ithas also become common to use the media to differentiate bacteria by their abilities toferment sugars other than lactose. In these cases lactose is replaced in the medium by anothersugar. These modified media are used to differentiate gram-negative bacteria or to distinguishbetween phenotypes with mutations that confer varying abilities to ferment particular sugars.

Theory

MacConkey agar is a selective and differential media used for the isolation anddifferentiation of non-fastidious gram-negative rods, particularly members of the familyEnterobacteriaceae and the genus Pseudomonas. The inclusion of crystal violet and bile saltsin the media prevent the growth of gram-positive bacteria and fastidious gram-negativebacteria, such as Neisseria and Pasteurella. The tolerance of gram-negative enteric bacteriato bile is partly a result of the relatively bile-resistant outer membrane, which hides the bile-sensitive cytoplasmic membrane (Nikaido, 1996). Other species specific bile-resistancemechanisms have also been identified (Provenzano, et al. 2000; Thanassi et al. 1997).

Gram-negative bacteria growing on the media are differentiated by their ability to ferment thesugar lactose. Bacteria that ferment lactose cause the pH of the media to drop and theresultant change in pH is detected by neutral red, which is red in color at pH's below 6.8. Asthe pH drops, neutral red is absorbed by the bacteria, which appear as bright pink to redcolonies on the agar.

The color of the medium surrounding Gram negative bacteria may also change. Stronglylactose fermenting bacteria produce sufficient acid to cause precipitation of the bile salts,resulting in a pink halo in the medium surrounding individual colonies or areas of confluentgrowth. Bacteria with weaker lactose fermentation growing on MacConkey agar will stillappear pink to red but will not be surrounded by a pink halo in the surrounding medium.

Gram-negative bacteria that grow on MacConkey agar but do not ferment lactose appearcolorless on the medium and the agar surrounding the bacteria remains relatively transparent.

Lactose can be replaced in the medium by other sugars and the abilities of gram-negativebacteria to ferment these replacement sugars is detectable in the same way as is lactosefermentation (for example Farmer and Davis, 1985).

RECIPE

Peptone (Difco) or Gelysate (BBL) 17.0 g

Proteose peptone (Difco) or Polypeptone(BBL) 3.0 g

Lactose 10.0 gNaCl 5.0 gCrystal Violet 1.0 mgNeutral Red 30.0 mgBile Salts 1.5 gAgar 13.5 gDistilled Water Add to make 1 Liter

Adjust pH to 7.1 +/-0.2. Boil to dissolve agar. Sterilize at 121° C for 15 minutes. (Holt andKrieg, 1994, Remel 2005)

PROTOCOL

Streak a plate of MacConkey's agar with the desired pure culture or mixed culture. If using amixed culture use a streak plate or spread plate to achieve colony isolation. Good colonyseparation will ensure the best differentiation of lactose fermenting and non-fermentingcolonies of bacteria.

Streak plate of Escherichia coli and Serratia marcescens on MacConkey agar. Bothmicroorganisms grow on this selective media because they are gram-negative non-fastidiousrods. Growth of E. coli, which ferments lactose, appears red/pink on the agar. Growth of S.marcescsens, which does not ferment lactose, appears colorless and translucent.

SAFETY

The ASM advocates that students must successfully demonstrate the ability to explain andpractice safe laboratory techniques. For more information, read the laboratory safety sectionof the ASM Curriculum Recommendations: Introductory Course in Microbiology and theGuidelines for Biosafety in Teaching Laboratories.

COMMENTS AND TIPS

This section is to evolve as feedback on the protocol is discussed at ASMCUE. Please contactthe project manager for further information.

REFERENCES

1. Difco Manual, Tenth Edition. 1984. Difco Laboratories, Inc. Detroit, MI., U.S.Grunbaum, A.S. and Hume, E. H. 1902. "Note on media for distinguishing B. coli, B.typhosus and related species." British Medical Journal, i: 1473-1474.2. Holt, J.G. and Krieg, N.R. 1994. "Chapter 8. Enrichment and Isolation." In [Eds.]Gerhardt, P., R.G.E. Murray, W.A. Wood and N.R. Krieg. Methods for General andMolecular Bacteriology. ASM Press, Washington, D.C. pg.2053. Collard, Patrick. 1976. "The Development of Microbiology". Cambridge UniversityPress, pp.31-32.4. Farmer JJ 3rd and Davis BR. 1985. "H7 antiserum-sorbitol fermentation medium: asingle tube screening medium for detecting Escherichia coli O157:H7 associated withhemorrhagic colitis." J Clin Microbiol. (4):620-5.5. Gerhardt, P., R.G.E. Murray, W.A. Wood and N.R. Krieg. Methods for General andMolecular Bacteriology. ASM Press, Washington, D.C. pg.2056. MacConkey, A. 1900. "A note on a new medium for the growth and differentiation of thebacillus Coli communis and the bacillus Typhi abdominalis." Lancet, ii:20.7. MacConkey, A. 1901. "Corrigendum et addendum." Zentralblatt fur Bakteriologie, 29:740.8. MacConkey, A. 1905. Lactose-fermenting bacteria in feces. J. Hyg.. 5:333-378.9. Nikaido, H. 1996. Outer membrane, p. 29-47. In F. C. Neidhardt, R. Curtiss III, J. L.Ingraham, E. C. C. Lin, K. B. Low, Jr., B. Magasanik, W. S. Reznikoff, M. Riley, M.Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella: cellular andmolecular biology, 2nd ed. ASM Press, Washington, D.C.10. Provenzano D, Schuhmacher DA, Barker JL, Klose KE. 2000 The virulenceregulatory protein ToxR mediates enhanced bile resistance in Vibrio cholerae and otherpathogenic Vibrio species. Infect Immun. 68(3):1491-711. Remel Microbiology Products. Instructions for Use of MacConkey Agar. Accessed June2005, http://www.remelinc.com/IFUs/IFU1550.pdf12. Ryan, K.J. and C.G. Ray (Ed.). 2004. Sherris Medical Microbiology. An Introductionto Infectious Disease. 4th Edition. McGraw-Hill, New York City, U.S.13. Thanassi, D. G., L. W. Cheng, and H. Nikaido. 1997. Active efflux of bile salts byEscherichia coli. J. Bacteriol. 179:2512-2518

REVIEWERS

This resource was peer-reviewed at ASM Conference for Undergraduate Educators 2005.

Participating reviewers:

Jay Mellies, Reed College, Portland, ORAnne Hanson, University of Maine, Orono, MEPatricia Shields, University of Maryland, College Park, MDDon Lehman, University of Delaware, Newark DELaboratory Protocols: http://www.microbelibrary.org/about/51

Blood Agar Plates and Hemolysis Protocols

Rebecca Buxton, 2013. Blood Agar Plates and Hemolysis Protocols. Visual Resources.American Society for Microbiology, Washington,DC. http://www.microbelibrary.org/component/resource/laboratory-test/2885-blood-agar-plates-and-hemolysis-protocols (Created: Friday, 30 September 2005, Last update: Monday,01 April 2013), RETRIEVED ON 25/06/2013

History

The history of blood agar, as we know it today, is uncertain. The inclusion of blood as anutritive supplement in culture media may pre-date the use of agar. In their 1903 Manual ofBacteriology, Muir and Ritchie list its inclusion before they discuss “agar-agar” as areplacement for gelatin as a solidifying agent.

In the same discussion, however, they note that Robert Koch preferred plates poured bymixing bacterial inocula with melted gelatin rather than streaking material on the surface.Koch recommended media that were “firm, and where possible, …transparent…” It appearsthat pour plates were the standard procedure for many years due largely to problems withsurface contamination upon incubation. (It should be noted that, initially, agar “plates” were,indeed, sterilized flat glass plates, not Petrie dishes as we know today.)

An interesting method of adding blood to agar media is described in Bulloch’s 1938 TheHistory of Bacteriology:

Human blood or the blood of animals may be used. “Sloped tubes” of agar areemployed. …Purify a finger first with 1-1000 corrosive sublimate, dry, and then washwith absolute alcohol to remove the sublimate. Allow the alcohol to evaporate. Prickwith a needle sterilised by heat, and, catching a drop of blood in the loop of a sterileplatinum wire, smear it on the surface of the agar. The excess of the blood runs downand leaves a film on the surface. Cover the tubes with India-rubber caps, andincubate them for one to two days at 37°C before use, to make certain that they aresterile. Agar poured out in a thin layer in a Petri dish may be smeared with blood inthe same way and used for culture. In investigating the diseases of races other thanthe white, it appears advisable to use the blood of the race under investigation.

Any reader interested in the history of microbiology should explore Wolfgang Hesse’sbiographical sketch of his grandparents, Walther and Angelina (“Lina”) (below). Walther wasa protégé of Koch, and Angelina served as his assistant and illustrator. Sometime prior to theend of 1882, Walther was frustrated by the melting of his gelatin-coated culture tubes in thesummer heat. He quizzed Lina about her jellies and puddings which maintained their solidconsistency even at the warm temperatures. It seems that she had learned about the use ofagar from a former neighbor (who had emigrated from Java where agar was a common foodadditive). Although there is no written record of such, it could easily be imagined thatWalther stirred blood into his cooled, melted agar in the same way that Lina did her fruit andmeat juices!

(Rebecca Buxton with archival help from Jeff Karr)

ASM News article "Walther and Angelina Hesse - Early Contributors to Bacteriology" byWolfgang Hesse and translated by Dieter H. M. Groschel (1992).

Purpose

Blood Agar is a general purpose enriched medium often used to grow fastidious organismsand to differentiate bacteria based on their hemolytic properties.

Theory

"Blood Agar" is not a consistently defined medium. The term "blood agar" generally refers toan enriched base medium to which defibrinated mammalian blood has been added.

In the US "blood agar" is usually prepared from Tryptic Soy Agar or Columbia Agar basewith 5% Sheep blood. Rabbit or horse blood may be used for growth of NAD-requiringorganisms, such as Haemophilus species, but the hemolytic patterns may be inconsistent withthose on sheep blood. (Human blood is discouraged because of the increased possibility ofexposure to human blood-borne pathogens such as HIV or hepatitis.)

FIG. 1. Tryptic Soy Agar with and without sheep blood

RECIPE

One commonly used formula:

Soybean-Casein Digest Agar

(Also referred to as "Trypticase Soy Agar" or "Tryptic Soy Agar" or "TSA" or "Blood AgarBase")

Pancreatic digest of casein USP 15.0 gPapaic digest of soy meal USP 5.0 gNaCl 5.0 gAgar 15.0 gDistilled water 1,000 mlCombine the ingredients and adjust the pH to 7.3. Boil to dissolve the agar, and sterilize byautoclaving.

Blood Agar

To sterile Blood Agar Base which has been melted and cooled to 45 to 50°C, add 5%(vol/vol) sterile defibrinated blood that has been warmed to room temperature. Swirl the flaskto mix thoroughly, avoiding the formation of bubbles, and dispense into sterile plates,continuing to avoid bubbles and froth on the surface. (NOTE: Cooling the agar and warmingthe blood are essential steps in this procedure. Hot agar can damage red blood cells, and coldblood can cause the agar to gel before pouring.)

PROTOCOL

Interpretation* of Hemolysis on Blood Agar Plates(*) To read the hemolytic reaction on a blood agar plate, the plate must be held up to a lightsource and observed with the light coming from behind (transmitted light).

Beta hemolysis (b) is defined as complete or true lysis of red blood cells. A clear zone,approaching the color and transparency of the base medium, surrounds the colony. Manyspecies of bacteria produce toxic by-products that are capable of destroying red blood cells.

FIG. 2 FIG. 3

FIG. 4

FIG. 2. Beta hemolytic Streptococcus species, Streptococcus pyogenes (transmitted light)(Lancefield group A).

FIG. 3.Normal Upper respiratory flora mixed with beta-hemolytic Streptococcus species.(The presence of beta-hemolytic colonies indicates the possibility of Streptococcuspyogenes infection.)

FIG. 4. Same blood agar plate as Figure 2 demonstrating that the beta hemolysis ofStreptococcus pyogenes is so complete that print my be read through the resultingtransparent medium.

Some species produce multiple toxins or display varying degrees of beta hemolysis.

One example is the occasional strain of Streptococcus pyogenes which produces only ahemolysin that is oxygen labile (“Streptolysin O”). In other words, the hemolysin is onlyactive in conditions of low oxygen. Hemolysis can be demonstrated by a pour plate or agaroverlay technique, or incubating in an anaerobic environment. A very simple way ofproducing an anaerobic “pocket” on an agar plate is to “stab” the inoculating loop verticallyinto the agar after streaking the plate. (Most strains of Streptococcus pyogenes also producethe oxygen stable hemolysin “Streptolysin S” which produces lysis in ambient air, as inFigure 3 above.)

Fig. 5 Fig. 6

Fig. 5 and Fig. 6. Normal Upper respiratory flora mixed with Streptococcus pyogenesdemonstrating production of Streptolysin O. Beta hemolysis is only evident where the agarwas “stabbed”.

Another example is found in Streptococcus agalactiae (Lancefield group B) and Listeriamonocytogenes. For these species, the hemolysin may be very slowly produced or weaklyreactive. The visible hemolysis may be so subtle that it is only apparent directly beneath thecolony (rather than broadly diffused as in S. pyogenes, above). In order to visualize this veryweak reaction, the colony may be removed with an inoculating loop, allowing one to view thelysed cells directly below where the colony had been growing. (For further information onStreptococcus agalactiae and multiple hemolysins, see CAMP procedure.)

FIG. 7 FIG. 8

FIG. 9

FIG. 7. Streptococcus agalactiae (Lancefield group B) viewed with incident light: Noobvious hemolysis.

FIG. 8. Streptococcus agalactiae (Lancefield group B) viewed with transmitted light: Subtlehemolysis.

FIG. 9. Listeria monocytogenes, removing colonies to see the subtle pink hemolysis directlybeneath the colonies

Alpha hemolysis (a) is the reduction of the red blood cell hemoglobin to methemoglobin inthe medium surrounding the colony. This causes a green or brown discoloration in themedium. The color can be equated with "bruising" the cells. Microscopic inspection of alpha-hemolyzed red blood cells shows that the cell membrane is intact, so it is not, in fact, truelysis. Some text book authors refer to alpha as “partial hemolysis,” which may be confusingto the student. It is most important to not confuse this “partial” or “incomplete” hemolysiswith the “weak” or “subtle” lysis of Streptococcus agalactiae or Listeria monocytogenes, asseen above. Beta hemolysis will never include the brown or green discoloration of the cells inthe surrounding medium. On prolonged incubation, many alpha hemolytic organisms willbegin to appear more clear, but if the surrounding medium contains any shades of brown orgreen the “hemolysis” is still considered “alpha.”

FIG. 10 FIG. 11

FIG. 10. Alpha-hemolytic Streptococcus species “Viridans group” streptococci, includingspecies such as the Streptococcus mutans, mitis, and salivarius groups display alphahemolysis.

FIG. 11. Alpha hemolysis of Streptococcus pneumoniae (Encapsulated strain).

Gamma hemolysis (g ) is somewhat self-contradictory. Gamma indicates the lack ofhemolysis. There should be no reaction in the surrounding medium.

FIG. 12 FIG. 13

FIG. 12. "Gamma Streptococcus" or Enterococcus faecalis (24 hours, non-hemolytic)."Gamma streptococcus" are usually non-hemolytic after 24 hours of incubation, but manyeventually display weak alpha hemolysis. (The genus Enterococcus was once a part of theStreptococcus genus, and was considered a "gamma Streptococcus species". Enterococciusually reacts as Lancefield group D.)

FIG. 13. The same Enterococcus strain as Figure (12), shown with transmitted light at 48hours incubation demonstrates the alpha hemolysis of some “gamma streptococci.”

SAFETY

The ASM advocates that students must successfully demonstrate the ability to explain andpractice safe laboratory techniques. For more information, read the laboratory safety sectionof the ASM Curriculum Recommendations: Introductory Course in Microbiology and theGuidelines for Biosafety in Teaching Laboratories.

COMMENTS AND TIPS

This section is to evolve as feedback on the protocol is discussed at ASMCUE. Please contactthe project manager for further information.

REFERENCES

For Recipes and Protocol:

1. Gerhardt, Philipp, R. G. E. Murray, Willis A, Wood, Noel R. Krieg. 1994.Methods for General and Molecular Bacteriology. American Society forMicrobiology, Washington, D. C. (p. 619, 642, 647).

2. Difco Manual. 1984. Dehydrated culture media and reagents for microbiology, 10thed., Difco Laboratories, Detroit.

For History:

1. Muir, Robert and James Ritchie. 1903. Manual of Bacteriology. The MacMillanCompany, London. (p. 226-229)

2. Bulloch, William. 1938. The History of Bacteriology. Oxford University Press,London. (p. 42).

3. Hesse, Wolfgang. 1992. Walther and Angelina Hesse – Early Contributors toBacteriology. ASM News. 58: 425-428.

REVIEWERS

This resource was peer-reviewed at ASM Conference for Undergraduate Educators 2005.

Participating reviewers:

Samuel Fan, Bradley University, Peoria, ILAshalla Freeman, University of North Carolina, Chapel Hill, NCRoxana Hughes, UNT Biological Sciences, Denton, TXD. Sue Katz, Rogers State University, Claremore, OKLucy Kluckhohn Jones, Santa Monica College, Santa Monica, CAPatricia Shields, University of Maryland, College Park, MDErica Suchman, Colorado State University, Ft. Collins, CO

Mueller-Hinton agar

Müller-Hinton agar is a microbiological growth medium that is commonly used forantibiotic susceptibility testing. It is also used to isolate and maintain Neisseria andMoraxella species.

It typically contains (w/v):[1]

30.0% beef infusion 1.75% casein hydrolysate 0.15% starch 1.7% agar pH adjusted to neutral at 25 °C.

Five percent sheep blood may also be added when susceptibility testing is done onStreptococcus species. This type is also commonly used for susceptibility testing ofCampylobacter.

It has a few properties that make it excellent for antibiotic use. First of all, it is a non-selective, non-differential medium. This means that almost all organisms plated on here willgrow. Additionally, it contains starch. Starch is known to absorb toxins released frombacteria, so that they cannot interfere with the antibiotics. Second, it is a loose agar. Thisallows for better diffusion of the antibiotics than most other plates. A better diffusion leads toa truer zone of inhibition.

1. Atlas, R.M. (2004). Handbook of Microbiological Media. London: CRC Press.p. 1226.

The Streak Plate Protocol

D. Sue Katz, 2013. The Streak Plate Protocol. Visual Resources. American Society forMicrobiology, Washington,DC. http://www.microbelibrary.org/component/resource/laboratory-test/3160-the-streak-plate-protocol (Created: Monday, 08 September 2008, Last update: Monday, 01 April 2013),retrieved on 25/06/2013.

History

The modern streak plate procedure has evolved from attempts by Robert Koch and otherearly microbiologists to obtain pure bacterial cultures in order to study them, as detailed in an1881 paper authored by Koch (5). Slices of sterilized potatoes became the first solid mediaemployed on which to grow bacteria. This process was a procedure that worked only for afew organisms and only until the bacteria decomposed the potato surface. A search for othermaterials led to experimentation with the suitability of gelatin and agar-agar as solidifyingagents. Gelatin was difficult to prepare and difficult to use at room temperature, let alone atthe higher temperature of an incubator, and many bacteria digest the protein. Agar, becauseof its characteristics of melting only when boiled, rarely being digested by bacteria, andproviding a substance in which other nutrients could be dissolved, proved to be a suitablematerial on which to grow bacteria. Agar was originally called agar-agar and is derived fromseaweed. The agar that we use today is the same substance as agar-agar, but it has beenprocessed by the manufacturer. Agar, as purchased 100 years ago, required filtering before itwas clear enough to use in media (12). In the early eras of microbiology, making media wasan extensive process of preparing the extracts of meat or other nutrient sources, as well aspurifying and filtering the gelatin or agar. Before the invention of the autoclave, sterilizingthe media properly was also time consuming. The 1939 edition of An introduction toLaboratory Technique in Bacteriology, an early microbiology lab manual, contains extensiveinstruction for students to prepare their own media from "scratch" (7) for use in thelab. Before R. J. Petri invented the petri dish, flat plates of glass covered by glass lids weremost commonly used to culture organisms in gelatin.

Even after agar was adopted and solid media were available, the streak plate was notcommonly used. Historically, microbiologists most frequently used pour plates to isolateorganisms for pure cultures. A pure culture was made from an isolated colony, representedonly one species or strain, and traditionally arose through the growth of a singlecell. Colonies are considered isolated if they are not touching any other colony. Isolatedcolonies were identified and transferred by streaking onto a new agar or gelatin plate using asterile needle, a process called "picking colonies." More rarely, a researcher would try toisolate organisms directly on the surface of a gelatin or agar plate. A typical description ofthe streaking process was given by Huber Williams, revised by Meade Bolton in A Manual ofBacteriology published in 1908 (11). "...the isolation of bacteria may sometimes be effectedby drawing a platinum wire containing material to be examined rapidly over the surface of apetri dish containing solid gelatin or agar; or over the surface of the slanted culture mediumin a test tube; or by drawing it over the surface of the medium in one test tube, then withoutsterilizing, over the surface of another, perhaps over several in succession."

Bacteriology textbooks and lab manuals from the early and mid 20th century did not mentionthe streak plate nor did they have our typical "isolation streak" exercise. For instance,isolation by streaking is absent from Buchanan and Buchanan, 1938 (2) and from Sherwood,Billings and Clawson's manual published in 1952 (10). During a literature search to pinpointthe first appearance of our modern streak plate, several papers published in the 1940s werefound to mention streak plates. However, these did not describe the process or illustrate theresults, and from the context, most probably referred to the process of picking colonies andcreating a pure culture in fresh media.

An early version of our modern isolation streak is found in Levine's An Introduction toLaboratory Technique in Bacteriology published in 1939 (7) and a similar version from 1954,in Salle's Laboratory Manual on Fundamental Principles of Bacteriology, 4th ed. (9). In thatprocess, the student picked up organisms on a needle or loop and then either stabbed into theagar or spread the loopful of the culture at the upper end of the petri dish to thin it out. Thena series of strokes 1/4-inch apart was made over the rest of the plate. Dr. Salle noted that thefirst streaks would contain too many organisms but that the last streaks should give isolatedcolonies. He suggested that a second plate be inoculated without flaming the wire loop first,to give a better chance of obtaining isolated colonies. This process dilutes the bacteria as theplate is streaked, similar to the dilution observed in a modern streak plate.

FIG. 1. An example of the one-directional streak pattern as described in the lab manuals byLevine and Salle (7, 9). The plate illustrated is a 100-mm petri dish.

In 1958, in the first edition of Laboratory Exercises in Microbiology, Pelczar and Reid (8)presented a streak plate exercise. It utilized a 4-quadrant streak pattern, and the proceduredescribed using both a loop and a needle in the streak and all streaks were in the samedirection, rather than both back and forth.

FIG. 2. A drawing representing the streak pattern recommended by Pelczar and Reid (8). Allstrokes of the loop or needle are done in a single forward direction, rather than in a back-and-forth pattern, as indicated by the arrowhead directions. The initial sector is at the top of theplate, followed clockwise by sectors 2, 3, and 4.

The earliest appearance of the three sector streak pattern (called the T streak) commonly usedtoday may be the 1961 photos published in Finegold and Sweeney (4). An illustrationdetailing how to perform this streak is in the 1968 edition of the Manual of BBL Products andLaboratory Procedures (1). In addition to the T streak, the BBL Manual illustrates two otherstreak patterns, neither of which is the simple monodirectional streak pattern used earlier inthe century.

Today, there are two most commonly used streak patterns, a three sector T streak and a fourquadrant streak. Microbiology lab manuals since the 1970s have presented an isolation streakexercise. Lab manual editions published between 1970 and 2000 illustrated and describedseveral streak pattern variations. However, today, almost all published microbiology labmanuals illustrate at least the T streak.

FIG. 3. A three sector T streak of Serratiamarcescens grown on trypticase soyagar. This illustrates a streak plate which hasmany isolated colonies.

FIG. 4. This plate illustrates a streak platewhich did not achieve isolation, and whichwould not be considered a good streak plateexample. This photograph is by Dr. Min-KenLiao, Furman University.

Purpose

The purpose of the streak plate is to obtain isolated colonies from an inoculum by creatingareas of increasing dilution on a single plate. Isolated colonies represent a clone of cells,being derived from a single precursor cell. When culture media is inoculated using a singleisolated colony, the resulting culture grows from that single clone. Historically, mostmicrobiology research and microbial characterization has been done with pure cultures.

Theory

One bacterial cell will create a colony as it multiplies. The streak process is intended tocreate a region where the bacteria are so dilute that when each bacterium touches the surfaceof the agar, it is far enough away from other cells so that an isolated colony can develop. Inthis manner, spreading an inoculum with multiple organisms will result in isolation of thedifferent organisms.

PROTOCOL

Mesophilic bacteria are generally streaked onto media solidified with 1.5% agar oragarose. Gelatin can be used if a high enough concentration of gelatin protein or a lowenough incubation temperature is used. Thermophiles and hyperthermophiles can also bestreaked onto growth media solidified with agar substitutes, such as Gelrite and guar gum.

One-hundred-mm-diameter petri dishes are the most commonly used size of plate forstreaking. The agar surface of the plate should be dry without visible moisture such ascondensation drops. Traditionally, inoculated petri dishes are incubated with the agar side upto prevent condensed moisture from falling onto the agar surface, which would ruin theisolation by allowing bacteria to move across the moist surface creating areas of confluentgrowth instead.

The inoculum for a streak plate could come from any type of source, for example clinicalspecimen, sedimented urine, environmental swab, broth, or solid culture. The two mostcommon streak patterns are the three sector T streak and the four sector quadrant streak.

In a streak plate, dilution is achieved by first spreading the specimen over the agar surface ofone sector. If a cotton swab or disposable loop or needle was used to inoculate the firstsector, it is now discarded into an appropriate container, while reusable loops, usually withnichrome or platinum wire (24 gauge), are flamed to incinerate any organisms on theloop. When cooled, the sterile loop is streaked through the initial sector and organisms arecarried into the second sector where they are spread using a zig-zag movement. In a similarmanner, the organisms present on the loop are incinerated after the second sector is streaked,and the third sector is streaked. For a four quadrant plate, the process is carried an extra step.

Detailed procedure for a Three Sector Streak, the T Streak: Reference J. Lammert,Techniques in Microbiology, A Student Handbook (6)

Materials:

Specimen to be streaked; this protocol is written for a test tube culture Transfer loop (usually nichrome, a nickel-chromium alloy, or platinum; it may also be

a single-use disposable plastic loop, which would be discarded between sectors ratherthan resterilized)

Bunsen burner Sterile petri dish with appropriate bacterial media, such as trypticase soy agar Labeling pen Sterile cotton swabs (if necessary to remove condensation from the agar surface and

from around the inner rim of the petri dish)

A. Label a petri dish.

Petri dishes are labeled on the bottom rather than on the lid. In order to preserve area toobserve the plate after it has incubated, write close to the edge of the bottom of theplate. Labels usually include the organism name, type of agar, date, and the plater's name orinitials. Using sterile cotton swabs, remove any visible water on the agar in the plate oraround the inner rim of the petri plate. Observe the plate and mentally divide it into threesectors, a "T." The area above the "T" will become the first sector streaked. The plate willthen be turned clockwise (if you are right handed) with the agar side up. The second sectorwill then be at the top for streaking and then the plate is turned again so that the third sectorcan be streaked.

B. Sterilize the transfer loop before obtaining a specimen.

In order to streak a specimen from a culture tube, metal transfer loops are first flamed so thatthe entire wire is red-hot. The incineration and flaming process is described below in theTips section. When flaming, the wire loop is held in the light blue area of a bunsen burnerjust above the tip of inner flame of the flame until it is red-hot. If a hot incinerator isavailable, the loop may be sterilized by holding it inside the incinerator for 5 to 7seconds. Once sterile, the loop is allowed to cool by holding it still. Do not wave it aroundto cool it or blow on it. When manipulating bacteria, transfer loops are usually held like apencil. If plastic disposable loops are being utilized, they are removed from the packaging toavoid contamination and after being used, are discarded into an appropriate container. A newloop is recommended for each sector of an isolation streak plate.

C. Open the culture and collect a sample of specimen using the sterile loop.

Isolation can be obtained from any of a variety of specimens. This protocol describes the useof a mixed broth culture, where the culture contains several different bacterial species orstrains. The specimen streaked on a plate could come in a variety of forms, such as solidsamples, liquid samples, and cotton or foam swabs. Material containing possibly infectiousagents should be handled appropriately in the lab (see biosafety references below), only bystudents with appropriate levels of skill and expertise.

Remove the test tube cap. It is recommended that the cap be kept in your right hand (thehand holding the sterile loop). Curl the little finger of your right hand around the cap to holdit or hold it between the little finger and third finger from the back. See theillustration. Modern test tube caps extend over the top of the test tube, keeping the rim of the

test tube sterile while the rim of the cap has not been exposed to the bacteria. The cap canalso be placed on the disinfected table, if the test tube is held at an angle so that aircontamination does not fall down into the tube and the test tube cap is set with the sterile rimon the table.

Insert the loop into the culture tube and remove a loopful of broth.

Replace the cap of the test tube and put it back into the test tube rack.

D. Streak the plate.

Inoculating the agar means that the lid will have to be opened. Minimize the amount of agarand the length of time the agar is exposed to the environment during the streak process.

1) Streak the first sector.

Raise the petri dish lid to insert the loop. Touch the loop to the agar area on the opposite sideof the dish, the first sector. Bacteria on the loop will be transferred to the agar. Spread thebacteria in the first sector of the petri dish by moving the loop in a back and forth manneracross the dish, a zig-zag motion. Make the loop movements close together and cover theentire first region. The loop should glide over the surface of the agar; take care not to dig intothe agar.

2) Between sectors.

Remove the loop from the petri dish and obtain a sterile loop before continuing to the secondsector. Either incinerate the material on the loop or obtain a sterile loop if using plasticdisposable loops. The loop must be cool before streaking can continue. Metal loops can betouched to an uninoculated area of agar to test whether they are adequately cooled. If theloop is cool, there will be no sizzling or hissing and the agar will not be melted to form abrand. If a brand is formed, avoid that area when continuing with the streaking process.

3) Streak the second sector.

Open the petri dish and insert the loop. Touch the cooled loop to the first sector once,invisibly drawing a few of the bacteria from the first sector into the second sector. Thesecond sector is streaked less heavily than the first sector, again using a zig-zag motion.

4) Obtain a sterile loop for the third sector (see 2, above).

5) Streak the third sector.

Open the petri dish and insert the loop. Touch the cooled loop (if the loop has been flamed)once into the second sector and draw bacteria from the second sector into the thirdsector. Streak the third sector with a zig-zag motion. This last sector has the widest gapbetween the rows of streaking, placing the bacteria a little further apart than in the previoustwo sectors. Watch closely to avoid touching the first sector as the streak is completed.

6) Final step.

Flame the loop to incinerate any bacteria that are left on the loop. Allow the loop to coolbefore placing it near anything that is flammable. Invert the petri dish so that the agar side isup and incubate the plates.

SAFETY

The ASM advocates that students must successfully demonstrate the ability to explain andpractice safe laboratory techniques. For more information, read the laboratory safety sectionof the ASM Curriculum Recommendations: Introductory Course in Microbiology and theGuidelines for Biosafety in Teaching Laboratories.

COMMENTS AND TIPS

A. Alternate streak patterns and different culture media

A variety of alternate streak patterns exist. Some are used for specific inocula, such as aurine specimen. The patterns also differ in the number of sectors as well as in the number oftimes the loop is sterilized.

The ur quadrant streak pattern would be recommended for use when large amounts ofbacteria are expected in the inoculum. The extra sector will provide additional dilution andincrease the probability of isolated colonies on the plate. The four quadrant streak plate isdescribed in a variety of references, e.g., in Cappuccino and Sherman's Microbiology, ALaboratory Manual, 8th ed. (3).

Sometimes, cultures will be streaked on enrichment media or various selective anddifferential media. For instance, a culture which is expected to have a gram-negativepathogen will be streaked on a MacConkey agar plate, which inhibits the growth of gram-positive organisms.

B. Incinerating material on transfer loops—flaming

Reusable microbiological loops and needles are sterilized by flaming. A Bunsen burner istraditionally used for this process. Most microbiology manuals show the microbiologistpositioned with his/her hand above the burner, with the loop placed into the flame. To avoidpossible contact with the flame, the microbiologist might consider placing his/her hand belowthe flame with the loop/needle above the hand in the flame. The flame of the Bunsen burnershould be adjusted to blue, with the darker blue cone of cooler air visible in the center of theflame. The loop or needle should be placed into the hotter part of the flame and kept thereuntil it glows red. There is a possible aerosolization hazard if the loop or needle containsliquid or a bacterial clump. These loops and needles should be placed into the heat slowly sothat the moisture evaporates rather than sputters.

If an incinerator such as a Bacti-Cinerator is used to sterilize the loop, the loop is to remaininside the incinerator for 5 to 7 seconds. When warmed up (which will take 5 minutes), thetemperature inside the incinerator is 815°C. The incinerator will take 5 to 10 minutes towarm up to working temperature.

C. Several techniques decontaminate transfer loops between sectors of a streak plate:flame, dig into agar, flame once and rotate loop

A variety of methods exist for removing organisms from the loop betweensectors. Beginning students are generally taught to sterilize the loop between each sector byincinerating and then cooling the loop. Clinical microbiologists practice a variety ofmethods. Some flame once after the initial sector and then rotate the loop so that the nextsectors can be streaked with an unused side of the loop. Other laboratorians (as clinicalmicrobiologists name themselves) stab the loop several times into the agar to clear the loopbetween sectors.

D. Isolated colony appearances

Isolated colonies can be described using the traditional colony descriptions. The ColonyMorphology Atlas-Protocol project provides information about bacterial colony appearanceand characteristic photographs. The appearance of an organism can vary. For instance, acolony of an organism growing in a crowded sector of the plate will not grow as large as theidentical organism growing in isolation. The media composition, pH, and moistness, as wellas the length of time and temperature can all affect the organism's appearance. Coloniesselected for subculturing should be colonies which are isolated, i.e., there is no other colonyvisibly touching the colony.

Agar with a surface layer of water is not suitable for obtaining isolated colonies. Obviouswater drops should be removed from the surface of the plate and from the rim of the plate byusing sterile cotton swabs. Plates should be incubated agar side up, to avoid condensationthat would drop onto the growing colonies on the agar surface.

E. Flaming tube mouths

Many protocols suggest flaming the tube mouth before and after removing organisms from atube. Flaming was important when test tubes were capped with a cotton plug. Flamingwould still be appropriate if a foam plug were being used. If a screw cap, KimKap, orsimilar test tube cap is used, the open end of the tube remains sterile since the cap normallycovers that area.

F. Rehearsing the streak procedure

Some instructors have students practice the streaking procedure on a piece of paper. Theprocess helps the student visualize the completed product and practice the fine musclemovements that are required in successful streaking for isolation.

Students may also find that they can visualize the pattern better if they mark the back of thepetri dish (for instance, a T streak divide the plate into three sectors).

Before learning to streak, students should have had the opportunity to work with 1.5% agarmedia. Ideally they will have also previously had the opportunity to practice using a loop ona plate to determine the best angle of approach and the amount of force required to glide theloop over the surface of the agar without gouging the surface.

G. Holding the plate while streaking

If possible, adequate lighting should be available to help the microbiologist follow thetracings of the loop on the agar. For most labs, this means that the petri dish should be heldin one's hand while being streaked in order to reflect the light properly. Additionally, thelength of time the petri dish lid is removed should be minimized in order to limitcontamination. There are several ways to hold the petri dish. Beginning students may findthat they obtain the best results leaving the plate on the lab bench and lifting the lid towork. Other students may find that they can place the plate upside down on the workbenchand lift the agar containing bottom, hold it to streak and then quickly replace it into thelid. Yet other students may have the manual dexterity to manipulate the entire dish in theirhand, raising the lid with thumb and two fingers while balancing the plate in the rest of theirhand.

REFERENCES

1. BBL. 1973. BBL manual of products and laboratory procedures. Becton DicksonMicrobiology Systems, Cockeysville, MD.2. Buchanan, E. D., and R. E. Buchanan. 1938. Bacteriology for students in general andhousehold science, 4th ed. Macmillan Company, New York, NY.3. Cappuccino, J. G., and N. Sherman. 2008. Microbiology a laboratory manual, 8thed. Pearson/Benjamin Cummings, San Francisco, CA.4. Finegold, S. M., and E. E. Sweeney. 1960. New selective and differential medium forcoagulase-positive staphylococci allowing rapid growth and strain differentiation. J.Bacteriol. 81(4):636–641.6. Lammert, J. M. 2007. Techniques in microbiology. A studenthandbook. Pearson/Prentice Hall, Upper Saddle River, NJ.7. Levine, M. 1939. An introduction to laboratory technique in bacteriology, reviseded. The Macmillan Company, New York, NY.8. Pelczar, M. J., Jr., and R. D. Reid. 1958. Laboratory exercises in microbiology, p. 45–47. McGraw-Hill Book Company, Inc., New York, NY.9. Salle, A. J. 1954. Laboratory manual on fundamental principles of bacteriology, 4th ed.,p. 39. McGraw-Hill Book Company, Inc., New York, NY.10. Sherwood, N. P., F. H. Billings, and B. J. Clawson. 1992. Laboratory exercises inbacteriology and diagnostic methods, 7th ed. The World Co., Lawrence, KS.11. Williams, H. U. 1908. A manual of bacteriology, p. 100. Revised by B. M. Bolton. P.Blakiston's Son & Co., PA.12. Williams, C. L., and H. P. Letton. 1916. A note on the preparation of agar agar culturemedia. J. Bacteriol. 1:547-548.http://jb.asm.org/cgi/reprint/1/5/547?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&author1=williams&author2=letton&titleabstract=agar-agar&searchid=1&FIRSTINDEX=0&tdate=3/31/1931&resourcetype=HWCIT.

REVIEWERS

This resource was peer-reviewed at the ASM Conference for Undergraduate Educators 2008.

Participating reviewers:

Gary Alderson, Palomar College, San Marcos, CAGail Baker, Okaloosa-Walton College, Niceville, FL

Kris Baumgarten, Delta College, University Center, MICristin Berkey, Children’s Hospital, Boston, MACarolyn Bouma, West Texas A & M University, CanyonMary Boyle, Vermont Technical College, Randolph CenterRebecca Buxton, University of Utah, Salt Lake CityLaura Cathcart, University of Maryland, College ParkKaren Dalton, The Community College of Baltimore County, Catonsville, MDDeanna Denault, River Valley Community College, Claremont, NHHeather Dietz, University of Regina, Saskatchewan, CanadaEllen Duffy, Albany College of Pharmacy, Albany, NYMichelle Furlong, Clayton State University, Morrow, GAMichaela Gazdik, Ferrum College, Ferrum, VAJanyce House, West Central Technical College, Waco, GAVicki Huffman, Potomac State College, Keyser, WVPhyllis Ingham, West Central Technical College, Waco, GAGary Kaiser, The Community College of Baltimore County, Catonsville, MDSamantha Kerry, St. Mary’s College of Maryland, St. Mary’s City, MDMin-Ken Liao, Furman University, Greenville, SCJohana Melendez-Santiago, Hillsborough Community College, Tampa, FLAmy Miller, Raymond Walter College, University of Cincinnati, Cincinnati, OHAngela NewMyer, St. Louis Community College at Forest Park, St. Louis, MOLaura Regassa, Georgia Southern University, Statesboro, GAJanet Rinehart-Kim, Tidewater Community College, Norfolk, VASeth Ririe, Brigham Young University–Idaho, Rexburg, IDBecky Sparks-Thissen, Wabash College, Crawfordsville, INSherrie Sprangers, University of Maine, Machias, MENick Tex, Apollo College, Phoenix, AZAnn Williams, Valrico, FL

Serial Dilution Protocols

Jackie Reynolds, 2013. Serial Dilution Protocols. Visual Resources. American Society forMicrobiology, Washington,DC. http://www.microbelibrary.org/component/resource/laboratory-test/2884-serial-dilution-protocols (Created: Friday, 30 September 2005, Last update: Monday, 01 April2013), retrieved on 25/06/2013.

It is a common practice to determine microbial counts for both liquid and solid specimens---suspensions of E. coli in nutrient broth all the way to soil samples and hamburger meat. Mostspecimens have high enough numbers of microorganisms that the specimen has to be seriallydiluted to quantitate effectively.The following is a step-by-step procedure to working dilutionproblems, and includes some practice problems at the end.

The purpose can be determination of bacterial, fungal, or viral counts. This protocol isspecific for bacterial counts (colony-forming units, CFUs), but can be modified for fungi(CFUs) and viruses (plaque-forming units, PFUs for viral counts).

History

Robert Koch is credited with identifying a method for bacterial enumeration, used first for the

study of water quality. His article, About Detection Methods for Microorganisms in Water.was published in 1883.

The standard plate count is a reliable method for enumerating bacteria and fungi. A set ofserial dilutions is made, a sample of each is placed into a liquefied agar medium, and themedium poured into a petri dish. The agar solidifies, with the bacterial cells locked inside ofthe agar. Colonies grow within the agar, as well as on top of the agar and below the agar(between the agar and the lower dish). The procedure described above produces a set of pourplates from many dilutions, but spread plates (sample spread on top of solidified agar) can beused also. The agar plate allows accurate counting of the microorganisms, resulting from theequal distribution across the agar plate. This cannot be done with a fluid solution since 1) onecannot identify purity of the specimen, and 2) there is no way to enumerate the cells in aliquid.

Principles

THE STANDARD FORMULA ( )( ( )To work the problem, you need 3 values---a colony count from the pour or spread plates, adilution factor for the dilution tube from which the countable agar plate comes, and thevolume of the dilution that was plated on the agar plate.

PROTOCOL

STEP 1: Determine the appropriate plate for counting:

Look at all plates and find the one with 30-300 colonies (see COMMENTS & TIPS section atend for explanation).

Use the total dilution for the tube from where the plate count was obtained.If duplicate plates (with same amount plated) have been made from one dilution, average thecounts together.

STEP 2: Determine the total dilution for the dilution tubes:

Dilution factor = amount of specimen transferred divided by the total volume aftertransfer[amount of specimen transferred + amount of diluent already in tube].

Determine the dilution factor for each tube in the dilution series.Multiply the individual dilution factor for the tube and all previous tubes.

To calculate this dilution series:

Determine the dilution factor of each tube in the set.

= +But after the first tube, each tube is a dilution of the previous dilution tube.

SO…..

total dilution factor = previous dilution factor of tube X dilution of next tube

FOR THE ABOVE DILUTION SERIES:

0.5 ml added to 4.5ml = 0.5/5.0 = 5/50 = 1/10 for 1st tube

1ml added to 9ml = 1/10 (2nd tube) X previous dilution of 1/10 (1st tube) = total dilution of1/100 for 2nd tube.

STEP 3: Determine the amount plated (the amount of dilution used to make the particularpour plate or spread plate).

There is nothing to calculate here: the value will be stated in the procedure, or it will be givenin the problem.

STEP 4: Solve the problem

1. The countable plate is the one with 51 colonies.2. The total dilution of the 2nd tube from which that pour plate was made = 1/102

3. The amount used to make that pour plate = 0.1ml (convert to 1/10 - it is easier tomultiply fractions and decimals together).

51 colonies = 51 X 103 = 5.1 X 104 (scientific notation) OR 51,000 CFUs/ml1/102 X 1/10

45 colonies = 45 X 104 = 4.5 X 105 (scientific notation) OR 450,000/ml1/103 X 1/10

SAFETY

Tubes and agar plates should be discarded properly in a biohazard container for propersterilization. The pipettes will also be sterilized (washed first if using reusable glass pipettes.

Do not pipette by mouth.

Use sterile technique in the transfer of microorganisms from tube to tube, as well as in theproduction of the pour plates.

The ASM advocates that students must successfully demonstrate the ability to explain andpractice safe laboratory techniques. For more information, read the laboratory safety sectionof the ASM Curriculum Recommendations: Introductory Course in Microbiology and theGuidelines for Biosafety in Teaching Laboratories.

COMMENTS AND TIPS

Greater than 300 colonies on the agar plate and less than 30 leads to a high degree of error.Air contaminants can contribute significantly to a really low count. A high count can beconfounded by error in counting too many small colonies, or difficulty in countingoverlapping colonies.

Use sterile pipettes for the dilutions, and use different ones in between the different dilutions.To do otherwise will increase the chances of inaccuracy because of carry-over of cells.

Accuracy in quantitation is determined by accurate pipette use and adequate agitation ofdilution tubes.

REFERENCES

There are no references at this time.

REVIEWERS

This resource was peer-reviewed at ASM Conference for Undergraduate Educators 2005.

Participating reviewers:

Donald Breakwell, Brigham Young University, Provo, UTJoan E. Cunnick, Iowa State University, Ames, IAMichelle Furlong, Clayton State University, Morrow, GAMark Gallo, Niagara University, Niagara University, NYChristopher Woolverton, Kent State University, Kent, OH