To Heat or Methanol Fix? That is the Burning Question.

Microbiologists are a very opinionated group of individuals, and it is, at times, difficult to get consensus and standardization on certain procedures. Whether to heat fix or methanol fix slides in preparation for Gram staining is one of those controversial procedures.technician reviewing slide

It was never fully documented as to why one method would be superior over the other… until now!

Jeanne Minnerath et. al. at the Biology Department at St. Mary’s University of Minnesota decided to settle the matter. They examined heat and methanol fixed slides for two parameters; adherence of the bacterial cells to the slide and the ability of the gram-positive bacteria not to become over decolorized.

Their results showed that methanol fixation won by a long shot!  There were 2.5 times as many Staphylococcus aureus cells present on the slide with methanol fixation compared to heat fixation. This number increased to a factor of 10 when E. coli was tested. The methanol fixation was superior in causing the specimen to adhere securely to the slide.

In addition, they found that Gram-positive bacteria were far less likely to become over-decolorized with methanol rather than heat fixation. This confirmed the findings of Mangels, et al in 1984.
Heat fixation tends to damage and distort the delicate cell wall structures of bacteria. This distortion also happens with tissue and blood cells which can create background debris that may be confusing or misleading.

Some have a concern for the toxicity and flammability of methanol in the lab. However, now that fume hoods are usually available and Bunsen burners are mostly used for museum displays, this should not be a problem. So why do some microbiologists insist on heat fixation?

Curiously, Minnerath mentions in her study that of the 15 microbiology manuals that they examined, all of them recommended heat fixation. Only two of them even mentioned methanol as an alternative.

Minnerath Study
Mangels Study

Some comments from a survey in October 2016 about Heat vs Methanol Fixation:

“Methanol fixation is superior, especially from blood culture bottles.”
Steven D. Dallas, PhD, D(ABMM), MT(ASCP)SM
Associate Professor, Departments of Clinical Laboratory Sciences and Pathology
UT Health Science Center San Antonio, Texas

“Jim Mangels convinced me back in the early 80s.  He was working at Good Samaritan at the time.
I think most hospitals in the Bay Area (I hope) use methanol.”
Bill Horbaly, O’Connor Hospital, California

“I’ve worked in a hospital since 1971 and in Microbiology since 1972. During the early 1980’s I attended a wet workshop on anaerobes. Our instructor recommended methanol fixing for morphology. Once back in my lab, I experimented by making two slides on various specimen types with one receiving heat fixing and the other methanol fixing.

There was definitely an adjustment when reviewing our slides. Once we instituted methanol fixing as our method of choice, we observed a variety of positive findings. There was less debris on our slides; the morphology was a little crisper; great capsules; we found Giardia on our stool gram stains when we were looking for WBC.

I taught Microbiology seminar classes for a Community College for 27 years starting in 1980. Some of these students also rotated through our hospital for their lab training. Initially, some of the students were conflicted because I was teaching something different from their Microbiology class at the college. Their instructor inquired about our method. I continued to use methanol fixing in my lab and taught both methods to the students with explanations as to the why. Several years ago, the college found the methanol mentioned in a text and moved forward to teach the students both methods.

I appreciate the studies confirming what I already knew.”
Carol Frantz, Microbiology, Milford Hospital, CT
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