From Polenta to Peptone

The Evolution of Bacterial Growth Media
 

by Ananya Sen
 

Figure 1. "Bloody bread": Serratia marcescens grow­ing on bread. CC BY-SA 3.0 Dbn

In the fall of 1918, the Italian chemist and pharmacist Bar­to­lo­meo Bizio studied the common occurrence of po­len­ta (corn meal mush) sometimes taking on a "brilliant red color." The red color was believed by the populace to be blood, and thus a miraculous portent. For many decades, farmers had lived in fear of this "bloody polenta" and would beseech their priests to exorcise the maleficent be­ings from their homes. Bizio, through a series of de­light­ful experiments, proved that the reddening was due to natural causes, when the air was damp and warm. Thus, the appearance of Serratia marcescens on polenta is an early recording of bacteria growing on solid surfaces.
 

However, culturing bacteria dates back to 1776 when Lazzaro Spallanzani, in his attempt to dis­prove the "spontaneous generation" of microbes, grew them in infusions of kidney beans, barley, maize, etc. The disadvantage of liquid cultures eventually became apparent ‒ they could not be used to make pure cultures of bacteria. Bacteria grew together, which made it impossible to dis­tin­guish between different kinds. Fifty years later Robert Koch laid the foundation for culturing bacteria on solid media. Koch attempted to isolate pathogens in pure culture by growing them on potato slices. This technique turned out to be unsuitable, as the slices would frequently become contaminated by fungi. Furthermore, potato slices had relatively few available nutrients and most bacteria did not grow well on them. As an improvement, Koch used gelatin as a solidifying agent. Although gelatin was effective, a major drawback was that it did not remain solid at 37°C (the op­timum growth temperature for most human pathogens) and would frustrate further studies of pure cultures. A better alternative was required.
 

Agar to the Rescue!

In 1881, Walther Hesse joined Koch's lab to study air quality. He trapped airborne microbes (using filters) and attempted to study them on potato slices or on gelatin-containing media. He faced the same technical challenges that plagued his colleagues. When Walther voiced his concerns to his wife and lab technician, New York City-born Fanny Hesse, she suggested the use of agar-agar. This was a common gelling agent in warmer countries, and a neighbor in her former hometown, who had immigrated from Java, had suggested it as a trick for solidifying jellies and thickening soups. This household hint transformed the technique of obtaining pure cultures. Agar is the perfect gel­ling agent ‒ it is solid at 37°C, not degraded by the great majority of bacteria, and can be easily poured into sterile containers.
 

Figure 2. A Bell jar (glass). Source. B Petri dishes. Source

The next question that arose was ‒ what sterile con­tain­ers to use? The state of the art system at the time was Koch's flat plate technique. This entailed pouring the agar based media into plates, stacking them, and covering them with a bell jar. Whenever the plates needed to be studied, the bell jar would be lifted, which would lead to frequent contamination. The final tweak in this system was made in 1887 by Richard Petri (also Koch's assistant). He designed the Petri dish which, as everyone knows, consists of two flat circular dishes where the upper dish serving as an overhanging lid. This simple modification has several advantages: the larger dish serves as a bar­ri­er against contamination, colonies on the surface of the plate are fully exposed to air and can be easily examined. Plus, the plates can be stacked and sterilized separately from the medium. With the help of the agar plates, microbiologists of the day (many of whom were Koch's students) went on to isolate the pathogens responsible for tuberculosis, diphtheria, typhoid, many pneumonias, meningitis, tetanus, and bubonic plague.
 

Selective Growth ‒ Survival of the Fittest

The next advancement was the development of selective media. Selective media favor the growth of certain bacteria based on specific metabolic characteristics, thereby allowing them to outgrow other bacteria. This technique is useful when bacteria must be isolated from natural environments where their numbers are too small to be studied directly. The development of selective media was pioneered by Sergei Winogradsky in 1888. He was studying sulfate reduction by Beggiatoa and was able to grow these organisms in pure culture from environmental samples. Almost si­mul­ta­ne­ous­ly, Martinus Beijerinck drew from Winogradsky's experiments and isolated nitrogen-fixing bacteria by growing them in a "poor nutrient medium at room temperature." Here, the only organisms that grew were those that could use atmospheric nitrogen. Beijerinck also used enrichment media to isolate a plethora of microorganisms with widely different metabolic properties ‒ sulfate-reducing bacteria, denitrifying bacteria, lactic acid, and acetic acid bacteria.
 

Differentiate and Conquer

Figure 3. Streptococcus agalactiae on Granada agar, anaerobic incubation. CC BY-SA 4.0 43trevenque

Isolating bacteria and growing pure cultures from en­vi­ron­men­tal samples was extremely helpful in cha­rac­te­riz­ing bacteria. However, closely related groups of bacteria could not be easily distinguished just using selective me­dia. To this end, Alfred MacConkey developed MacConkey medium in 1905. Among other ingredients, this medium contained bile salts (that inhibit certain groups of bac­te­ria) and a pH-sensitive dye, neutral red. The rationale was that if the sugars in the medium were broken down by the bacteria, the acid released would turn the dye red. If the bacteria were incapable of this reaction, the colonies would remain colorless. Other examples of differential media are EMB agar (Holt-Harris & Teague, 1916) that dif­ferentiates between lactose fermenters and lactose non-fermenters, mannitol salt agar (Chapman, 1945) that differentiates mannitol fermenting and mannitol non-fermenting species of Staphylococcus, and Granada medium (De La Rosa et al., 1983) that differentiates between hemolytic and non-hemolytic Group B Streptococcus strains.
 

Note on LB

LB is a widely used growth medium for many diverse studies with E. coli. Since many labs use it, I thought it would be interesting (while running the risk of appearing to be an E.coli snob) to learn a little more about this medium’s origins. It was developed in 1951 by Giuseppe Bertani who was studying lysogeny (hence Lysogeny Broth, or LB). The components of this medium are yeast ex­tract, sodium chloride and a peptone. Peptone is the generic term for products made by en­zy­ma­tic cleavage of protein. In the case of LB, the peptone that is used in called tryptone, the result of digesting the milk protein casein with the protease trypsin. Although it is unclear when the in­cor­po­ra­tion of yeast extract in media came to be, the addition of peptones to growth medium can be traced back to Friedrich Loeffler in 1887. Peptones are a rich source of amino acids and other ni­tro­genous compounds that helped the growth of Corynebacterium diphtheriae. In the same pa­per, he also discussed adding salt for osmotic balance. LB is now used as a standard medium in ma­ny different labs, from microbiology to biochemistry to cell biology (all thanks to LB's pre­do­mi­nant consumer and wonder bug, E. coli ).
 

Figure 4. LB medium: A liquid, B solid (agar) in Petri dish. GFDL Masur

A word of caution: LB does have several drawbacks ‒ nei­ther tryptone nor yeast extract have de­fined com­po­si­tions, causing batch-to-batch variability. Thus, the results in LB are often hard to re­produce accurately. In addition, it has only a low amount of fermentable carbohydrates (unless ad­ded), and is poor in magnesium. The minus si­d­es of LB have been neatly summarized by Hiroshi Ni­ka­i­do. To counter the things that are wrong with LB, Neid­hardt, Bloch, and Smith developed in 1974 a basic mi­ni­mal medium that can be enriched at will by the addition of other nutrients. This medium has defined components , thus makings its chemical composition reproducible without compromising the ability to supplement extra desired nutrients.Thus, it can be used to grow bac­te­ria at a variety of rates.
 

This is not the end of the story on microbial growth media. Development of new growth media to meet specific needs is a very important ongoing ef­fort in the microbial sciences. Witness, for ex­am­ple, the recent publication of a nearly universal me­dium that permits the growth of anaerobic bacteria in normal atmosphere conditions.
 

Microbial art

Having all these different media is well and good for advancing the march of science. But what about man's innate need to see art in everything? Marc Chagall once said "Great art picks up where nature ends." Painters of the microbiology sort use bacteria to make fine art. The canvases are a medley of agar plates onto which microbes have been 'painted.' From Van Gogh's Starry Night to a humble mug of beer, the range of microbial art can be found here. Enjoy the art and remember the centuries of work that led to it.
 

 

 

 

Ananya is a graduate student in the Department of Microbiology at the University of Illinois at Urbana-Champaign. She works in the lab of James A. Imlay. Ananya has recently started a blog of her own called “ The History of Science."