IntroductionThe identification of bacteria and other microbes is a core aspect ofmicrobiology, and allows us to research, diagnose, and treat a variety ofdiseases. A key general method for bacterial and microbe identification isstaining. Staining methods consist of two broad types of stains: simple anddifferential.
Both simple stains and differential stains are essential toidentifying bacteria, whether by adding contrast to better visualize thebacteria to identify them based on morphology, or by differential methods to identifybacteria similar in morphology from each other based on other essentialcharacteristics such as cell wall structure. Simple staining uses a single dye,with examples of common dyes including crystal violet, methylene blue,malachite green, and safranin (Anderson,Salm, Allen, & Nester, 2016, p. 54). In the case of a simple stain, the dye providescontrast to cells that would otherwise appear opaque, and thus gives moredetailed definition to their morphology. Differential stains are multi-step processes, and can be used toidentify different types of bacteria based on unique traits not visible bystandard light microscopy. One of the most common and medically importantmethods of differential staining is the Gram stain, which stains Gram-positivebacteria blue-purple and Gram-negative bacteria pink-red. The Gram stainseparates the two types of bacteria using differences in cell wall structure:the thick peptidoglycan of the Gram-positive cell wall dehydrates during analcohol rinse applied after a crystal violet stain and prior to a safraninstain, and the crystal violet remains locked inside these Gram-positive cells.
Asthe difference in cell wall structure of Gram-positive and Gram-negativebacteria is important in the selection of antibiotics, the Gram stain is oftenused to inform the choice of treatment for bacterial infections. Simple stains and differential stains are used for a wide variety ofunique purposes, and new applications of stains are frequently discovered toaid in the process of identification of microbes. In some cases, researchersare exploring simpler staining methods that may achieve faster orhigher-quality results than standard methods. In a paper on a modified versionof Field’s staining for identification of Trichomonas vaginalis, researchersdiscovered that a simple rapid stain using this method provided a preferablealternative to the two standard methods of Giemsa staining and Gram staining (Afzan, Sivanandam, & Kumar, 2010).
Stainingmethods aid in rapid identification of microbes as compared to otheridentification methods, and are often accessible in settings where complextesting is unavailable, for instance in rural settings and developing countries(Moemen, Bedir, Awad, & Ellayeh, 2015; Wang& Murdoch, 2004). The efficiency of identification via stainingmethods is also essential when a suspected infection requires rapid treatmentin order to achieve a favorable outcome for the patient (Moemen et al., 2015). Materials and Methods The goal of this lab was for students to conduct gram stains on threedifferent species of bacteria: Escheria coli, Staphylococcus saprophyticus, andBacillus subtilis. A previously scheduled lab relevant to this report onsimple staining procedure was not conducted.
Initially, six slides werewet-mounted (two of each sample), allowed to air-dry until no liquid wasvisible, and then heat fixed. Aseptic technique was used to transfer a singleloop of each species to the slides. After heat fixing, a gram stain of the E.coli sample was attempted, using the following standard process provided: · Crystal violet applied to flat slide(60 seconds)· Water rinse drop-wise (10 seconds)· Iodine applied to flat slide (60 seconds)· Gentle water rinse drop-wise (10seconds) · Decolorizing alcohol rinse drop-wise(10 seconds) · Gentle water rinse (10 seconds) · Safranin applied to flat slide (60seconds) · Gentle water rinse (10 seconds)Upon inspection of the initial E. coli slide, no bacteria couldbe visualized. Multiple people in the lab inspected the slide via microscope,and none could locate bacteria. This process was then repeated twice with the B.
subtilis species, and no bacteria could be visualized. Upon conferringbetween partners and instructors in the lab, it was decided that an inadequateamount of substance might have been added to the slides. During the first threegram stain attempts, it seemed that the visible sample was fully washed awayfrom the slides during the alcohol rinse. Thus, two changes were made toattempt a successful stain: the use of additional culture on the smear, and areduction in time and volume of the alcohol rinse. Two additional slides, oneof S. saprophyticus and one of E. coli, were wet-mounted with threeloops of sample and heat fixed. Following heat fixing, the revisedmethod of staining was used on each slide.
Results Of the five total gram stains attempted,bacteria were visualized only for a single slide of S. saprophyticus. Thiswas the fourth slide prepared, and the first prepared using the modifiedtechnique. These bacteria stained purple in color and were spherical in shape.The bacteria clustered together, primarily in chains but occasionally in largerclusters, as indicated in Photograph 1, below.The final slide was also prepared using the modified technique, butyielded no visible bacteria.
Table 1, below, summarizes the results of the fiveattempted stains. The deviations described were the only changes made betweenthe first three and the last two samples.Conclusion This lab highlighted that differential staining requires a high degreeof precision in the multi-step process. While we found a solution to ourinitial problem, we were unable to replicate the solution a second time.
Thereare numerous variables that could explain this. As we obtained a successfulstain of one species but not the others, some of the samples may have beensparse in terms of bacterial growth. However, due to the visibility of thesmear prior to the alcohol rinse and the full disappearance of the smearfollowing the alcohol rinse in each unsuccessful stain, this is not the mostlikely explanation. Human error due to a learning curve with the Gram stainprocedure is a more likely explanation, as is the possibility of an overlyconcentrated or otherwise incorrect alcohol solution. The results we did obtain were as expected. Staphylococcussaproyphyticus is a Gram-positive coccus, and thus we would expect it tostain blue-purple. While we were unable to successfully stain the otherbacterial samples, we would have expected Escheria coli, a Gram-negativebacterium, to be rod-shaped and stain pink, and the Bacillus subtilis, aGram-negative bacterium, to be rod-shaped and stain purple.
The mostmeaningful takeaway from this lab is that differential staining procedures maytake practice to execute. We also learned the importance of familiarity withand understanding of equipment and materials, as we left the lab still unsureof which type of alcohol we had used during the alcohol rinse step where our challengesarose.