- 00:01Concepts
- 03:09Media Preparation
- 05:12Preparing Agar Plates
- 06:21Culturing Host Cells
- 07:30Phage Serial Dilution and Preparation of Bacteria and Phage Overlay
- 10:37Data Analysis and Results
- 11:35Results
Ensaio de placa: um método para determinar o título viral como unidades formadoras de placa (PFU)
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Overview
Fonte: Tilde Andersson1, Rolf Lood1
1 Departamento de Ciências Clínicas Lund, Divisão de Medicina de Infecção, Centro Biomédico, Universidade de Lund, 221 00 Lund, Suécia
Vírus que infectam organismos procarióticos, chamados bacteriófagos ou simplesmente fálticos, foram identificados no início do séculoXX por Twort (1) e d’Hérelle (2) de forma independente. Desde então, os phages têm sido amplamente reconhecidos por seu valor terapêutico (3) e sua influência sobre os humanos (4), bem como os ecossistemas globais (5). As preocupações atuais têm alimentado um interesse renovado no uso de phages como alternativa aos antibióticos modernos no tratamento de doenças infecciosas (6). Essencialmente, todas as pesquisas de phage dependem da capacidade de purificar e quantificar vírus, também conhecido como um titer viral. Inicialmente descrito em 1952, este era o propósito do ensaio da placa (7). Décadas e múltiplos avanços tecnológicos depois, o ensaio da placa continua sendo um dos métodos mais confiáveis para a determinação do título viral (8).
Bacteriophages subsistem injetando seu material genético em células hospedeiras, sequestrando as máquinas para produção de novas partículas de phage, e eventualmente fazendo com que o hospedeiro libere numerosos virions progêneres através da lise celular. Devido ao seu tamanho minucioso, os bacteriófagos não podem ser observados usando apenas microscopia leve; portanto, é necessária a microscopia eletrônica de varredura (Figura 1). Além disso, os phages não podem ser cultivados em placas de ágar nutricional como bactérias, pois precisam de células hospedeiras para se aproveitarem.
Figura 1: A morfologia de um bacteriófago, aqui exemplificado por um phage E. coli, pode ser estudado usando microscopia eletrônica de varredura. A maioria dos bacteriófagos pertence a Caudovirales (bacteriófagos tailed). Este phage em particular tem uma estrutura de cauda muito curta e uma cabeça icosaedral, colocando-a na família de Podovírus.
O ensaio da placa (Figura 2) baseia-se na incorporação de células hospedeiras, preferencialmente em crescimento de fase de registro, no meio. Isso cria uma densa camada turva de bactérias capazes de sustentar o crescimento viral. Uma praga isolada pode infectar, replicar dentro e lise uma célula. A cada célula lise, várias células adjacentes ficam imediatamente infectadas. Vários ciclos dentro, uma zona clara (uma placa) pode ser observada na placa turva (Figura 2B/Figura 3A), indicando a presença do que inicialmente era uma única partícula bacteriófago. O número de unidades formadoras de placas por volume (ou seja, PFU/mL) de uma amostra, pode ser determinado a partir do número de placas geradas.
Figura 2: O teste para unidades de formação de placas (PFU) é um método comum para determinar o número de bacteriófagos em uma amostra. (A) A base de uma placa de Petri estéril é coberta com um meio de nutrientes sólidos apropriado, seguido por uma mistura de mídia macia, células hospedeiras suscetíveis e uma diluição da amostra original de bacteriófago. Note que a suspensão de phage poderia, em alguns casos, também ser uniformemente espalhada pela superfície de ágar macio já solidificado. (B) O crescimento das bactérias hospedeiras forma um gramado de células na camada superior do ágar. A replicação do bacteriófago gera zonas claras, ou placas, causadas pela lise celular hospedeira.
Figura 3: Os resultados do teste de PFU mostram múltiplas placas geradas por bacteriófagos. Devido à lise de células hospedeiras suscetíveis, as placas podem ser vistas como zonas de compensação no gramado bacteriano, seja com (A) desobstrução total, ou (B) re-crescimento parcial causado pela geração de bactérias resistentes (ou possivelmente por phages temperados no ciclo lisogênico).
Certos fáticos temperados podem adotar o que é chamado de ciclo de vida lisogênico, além do crescimento lítico descrito anteriormente. Na lysogenia, o vírus assume um estado latente através da incorporação de seu material genético no genoma da célula hospedeira (9), muitas vezes conferindo resistência a novas infecções por pragas. Isso às vezes é revelado através de uma leve nebulosidade da placa (Figura 3B). Vale ressaltar, porém, que as placas também podem aparecer borradas devido ao reesúde de bactérias que evoluíram resistência ao phage independente de infecções anteriores.
Os vírus podem se conectar, ou adsorb, a apenas uma gama limitada de bactérias hospedeiras (10). As faixas de host são ainda limitadas por estratégias antivirais intracelulares, como o sistema CRISPR-Cas (11). A resistência/sensibilidade para phages específicos exibidos por subgrupos bacterianos tem sido historicamente usada para categorizar cepas bacterianas em diferentes tipos de phage (Figura 4). Embora a eficácia deste método tenha sido agora superada por novas técnicas de sequenciamento, a digitação de phage ainda pode fornecer informações valiosas sobre interações bactérias-phage, por exemplo, facilitando o design de um coquetel phage para uso clínico.
Figura 4: Sensibilidade à praga de diferentes cepas bacterianas. Placas de ágar macio com cepa cutibacterium acnes (A) AD27 e (B) AD35, foram avistadas com 21 bacteriófagos C. acnes diferentes. Apenas o phage 11 foi capaz de infectar e matar AD27, enquanto a cepa AD35 mostrou sensibilidade para todas as pragas. Esta técnica, denominada digitação phage, pode ser usada para dividir espécies bacterianas e cepas em diferentes subgrupos com base na suscetibilidade de phage.
Procedure
Applications and Summary
Despite multiple technological advances, plaque assays remain the gold standard for determination of viral titer (as PFU) and essential for isolation of pure bacteriophage populations. Susceptible host cells are cultivated in the top coat of a two layered agar-plate, forming a homogenous bed enabling viral replication. The initial event where an isolated bacteriophage in lytic lifecycle infects a cell, replicates within it, and eventually lyses it, is too small to observe. However, the virions released will infect adjacent cells, subsequently giving rise to a clearing zone, or plaque, denoting the presence of a single PFU.
References
- Twort, F. An investigation on the nature of ultra-microscopic viruses. Lancet. 186 (4814): 1241-1243. (1915)
- d'Hérelle, F. An invisible antagonist microbe of dysentery bacillus. Comptes Rendus Hebdomadaires Des Seances De L Academie Des Sciences. 165: 373-375. (1917)
- Cisek AA, Dąbrowska I, Gregorczyk KP, Wyżewski Z. Phage Therapy in Bacterial Infections Treatment: One Hundred Years After the Discovery of Bacteriophages. Current Microbiology. 74 (2):277-283. (2017)
- Mirzaei MK, Maurice CF. Ménage à trois in the human gut: interactions between host, bacteria and phages. Nature Reviews Microbiology. 15 (7):397. (2017)
- Breitbart M, Bonnain C, Malki K, Sawaya NA. Phage puppet masters of the marine microbial realm. Nature Microbiology. 3 (7):754-766. (2018)
- Leung CY, Weitz JS. Modeling the synergistic elimination of bacteria by phage and the innate immune system. Journal of Theoretical Biology. 429:241-252. (2017)
- Dulbecco R. Production of Plaques in Monolayer Tissue Cultures by Single Particles of an Animal Virus. Proceedings of the National Academy of Sciences of the United States of America. 38 (8):747-752. (1952)
- Juarez D, Long KC, Aguilar P, Kochel TJ, Halsey ES. Assessment of plaque assay methods for alphaviruses. J Virol Methods. 187 (1):185-9. (2013)
- Clokie MRJ, Millard AD, Letarov AV, Heaphy S. 2011. Phages in nature. Bacteriophage. 1 (1):31-45. (2011)
- Moldovan R, Chapman-McQuiston E, Wu XL. On kinetics of phage adsorption. Biophys J. 93 (1):303-15. (2007)
- Garneau JE, Dupuis M-È, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S.. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 468 (7320):67. (2010)
Transcript
Bacteriophages, also called phages, are viruses that specifically infect bacteria and we can confirm their presence and quantify them using a tool called the plaque assay. Bacteriophages infect their susceptible hosts by first attaching to the bacterial cell wall and injecting their genetic material. Then, they hijack the cell’s biosynthetic machinery to replicate their DNA and produce numerous progeny phage particles, which they then release by lysing and killing the host cell.
This lytic activity is the basis of a widely used phage enumerating technique known as the plaque assay or double agar layer assay. Here, a bacteriophage mix is first prepared in a molten nutrient broth containing low concentration agar. All bacteria used in the mix should be alive and actively dividing in the log phase of their growth, which will ensure that a large percentage of the bacteria are viable and able to form a dense lawn around the plaques. Next, this molten bacterial-phage agar mix is spread over a more solid, concentrated agar nutrient medium which is already solidified on a Petri dish. On incubation at room temperature, the low concentration agar-phage-bacteria broth also solidifies to form a soft agar overlay.
Here, the bacterial cells can derive additional nutrients from the bottom layer and should rapidly multiply to produce a confluent lawn of bacteria. However, as phage particles are also present in the soft layer, these will infect and replicate their genetic material within the bacteria, culminating in cell lysis, which releases multiple progeny. These phage progeny then infect the neighboring cells, as the semi-solid state of the bacteria-phage layer restricts their movement to more distantly located host cells. This cycle of infection and lysis continues over multiple rounds, killing a large number of bacteria in a localized area. The effect of the neighboring cells being destroyed, is to produce a single circular clear zone, called a plaque, which can be seen by the naked eye, effectively amplifying the bacteria lytic activity of the phage and enabling their enumeration.
The number of plaques on a Petri dish are referred to as Plaque-Forming Units, or PFUs, and, providing the initial bacteriophage concentration was sufficiently dilute, should directly correspond to the number of infective phage particles in the original sample. This technique can also be used for characterization of plaque morphology, to aid in identification of phage types, or to isolate phage mutants. In this lab, you will learn how to perform the plaque assay for enumerating phages, using the T7 phage of E. coli as an example.
First, identify a suitable medium for the culturing of the host bacterial cells and the bacteriophage. Here lysogeny broth, or LB medium, was used to culture E. coli and the T7 phage. Next, take three clean glass bottles and label them with media, name, and then the first as LB-Broth, the second as LB-Bottom Agar, and the third as LB-Top Agar. Now, weigh out four grams of pre-formulated LB powder in three sets and then transfer one set of weighed dried media into each bottle. Add 200 milliliters of water to the first bottle. Mix the contents using a magnetic stir bar.
Then, using a pH meter and constant stirring, bring the final pH to 7.4 through the addition of sodium hydroxide or hydrochloric acid. Repeat the water addition and pH adjustment for the other two remaining bottles, as well. Now, weigh out three grams of agar powder and add it to the second bottle to make a 1.5 % bottom agar. Finally, weigh 1. 2 grams of agar and add it to the third bottle to make the .6 % LB top agar. The broth condition in bottle one does not need an agar addition. Cap the bottle semi-tightly and then, sterilize the media by autoclaving at 121 degrees Celsius for 20 minutes. Once complete, remove the media bottles from the autoclave and immediately twist the bottle caps to close them fully to prevent contamination. Keep the LB-Broth and LB-Top Agar media on the bench for later use. Place the LB-Bottom Agar to cool in a water bath that is preset to approximately 45 degrees Celsius.
When the LB-Bottom agar reaches approximately 45 degrees Celsius, transfer it to the work bench. Next, sterilize the workspace using 70 % ethenol. Next, add 450 microliters of sterile one molar calcium chloride to the molten bottom agar to make a final concentration of 2.25 millimolar. Gently swirl the bottle to mix. Then, set out seven clean Petri dishes. Label each dish on the bottom with the media name and preparation date. Then, pour 15 milliliters of the bottom agar into each of the seven Petri dishes. Allow the plates to set for a few hours or overnight at room temperature. Once set, the culture plates can be stored at four degrees Celsius for several days if needed, upside down to minimize condensation. Transfer the Petri dishes from the four degrees Celsius refrigerator to a 37 degrees Celsius incubator one hour before the assay.
The day before the assay is to be preformed, the E. coli should be cultured. Here, 10 microliters of E. coli culture was inoculated into 10 milliliters of LB-Broth. Place the bacteria to grow overnight in a shaking incubator set to 37 degrees Celsius at 160 RPM. Then, on the day of the assay, remove the bacterial culture from the incubator. Seed a fresh 10 milliliters of fresh LB broth with 0.5 milliliters of the overnight culture. Place these cells to grow into a shaking incubator set to 37 degrees Celsius at 160 RPM. Next, use a spectrophotometer to check when this culture reaches log phase growth, indicated by an optical density of 0.5 to 0.7. Once the OD reaches this level, stop the incubation by transferring the cell culture to the bench. They are now ready to be used for phage overlay assay.
Phage titers can vary exponentially across different phage types and samples. So in order to count them effectively, they should be diluted to generate a wide range of phage concentrations. On the day of the assay, generate a series of phage dilutions ranging from one tenth to one millionth concentrations, following a 10-fold dilution technique. To obtain statistically significant and accurate data, perform the serial dilution in triplicate.
Next, melt the solidified LB-top agar using a microwave. Then, place it in a water bath that is preset at 45 degrees Celsius for one hour. After one hour, collect the Petri dishes containing the bottom agar layer from the incubator. Label the plates with phage concentration and assay date. Then, set out seven clean test tubes. Label each test tube with the serial phage dilution number and designate one as control.
When the LB-top agar reaches 45 degrees Celsius, transfer it to the working bench. Now, add 450 microliters of one molar calcium chloride to the 200 milliliter agar to make a final concentration of 2.25 millimolar. Gently swirl the bottle to mix. Next, add 35 milliliters of LB-top agar and four milliliters of bacterial suspension to a sterile conical tube. Gently swirl to evenly distribute the cells but avoid shaking to prevent foaming.
Now, aliquot five milliliters of this bacteria- top agar mix to each of the seven test tubes. Then, transfer 100 microliters of the serially diluted bacteriophage samples and control media, which should be simply media with no bacteriophage, to the respectfully labeled test tubes. Swirl the mixture gently to ensure proper mixing. Gently transfer five milliliters of bacteriophage mix onto the respective Petri plate. Evenly spread the mix throughout the whole surface by gently swirling the Petri plate.
Once all of the Petri plates are layered with the mix, allow solidification of the top layer by incubating at room temperature for 15 minutes. After completion of these steps, repeat the process for the second and then the third sets of the Petri dishes using the remaining two sets of phage dilutions. Seal each dish with parafilm and incubate at room temperature for 15 minutes. Place the culture plate upside down at a suitable temperature for 24 hours or until plaques develop. Here, plates were placed in a 37 degrees Celsius incubator for one day, a stimulating growth condition for E. coli and the T7 phage.
Plaques will appear after one to five days of incubation, depending on the bacterial species, incubation conditions, and the choice of medium. Here, plaques were visible after one day of incubation at 37 degrees Celsius. Begin by checking the plates marked control and ensure that no plaques were formed in these plates, as this would indicate viral contamination. To determine the phage titer in the original sample, start with the plates containing the most diluted phage sample first and count the plaques without removing the lids, marking them to indicate which ones have already been counted. Repeat the counting for each plate in every set. Some plates might have too many or too few plaques to be counted. Consider 10 to 150 as an ideal plaque count.
Next, generate a table listing the plaque number values for the different dilutions and replicates. Then, calculate the mean plaque number values for the dilution plates that contained the ideal number of plaque counts. In this example, these were the average number of plaques formed in the 10 to the minus three and 10 to the minus four dilution plates. Now, adjust for phage dilution factor by dividing the obtained mean plaque values by the respective phage dilution factors. Here, the average number of plaques formed to the 10 to the minus three and 10 to the minus four dilution plates, were divided by their respective dilution factors to obtain the number of plaque forming units, or PFUs, in 100 microliters of phage mixture. To convert the value to PFU per milliliter, multiply the generated values by 10, as only 100 microliters of phage dilution mix was used during the bacteriophage overlay preparation step, producing an additional dilution factor of 10. Finally, calculate the average of the values obtained from the different dilution plates. This will give the average number of PFUs per milliliter. The number of PFUs corresponds to the number of infective phage particles in the original sample.